ELPS elps201200082 Dispatch: May 11, 2012 CE: Journal MSP No. No. of pages: 5 PE: XXXXX 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 UNCORRECTED PROOF Electrophoresis 2012, 33, 1–5 1 Nadia Cufos 1,2* Abdul Jabbar 1* Lu´ ıs M. de Carvalho 2 Robin B. Gasser 1 1 Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia 2 Interdisciplinary Research Centre on Animal Health, Faculty of Veterinary Medicine, Technical University of Lisbon (CIISA/FVM/TUL), P ´ olo Universit ´ ario do Alto da Ajuda, Lisbon, Portugal Received February 7, 2012 Revised March 18, 2012 Accepted March 19, 2012 Research Article Mutation scanning based analysis of Theileria orientalis populations in cattle following an outbreak Bovine theileriosis is a tick-borne disease caused by one or more hemoprotozoan parasites of the genus Theileria. In the past, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli. However, outbreaks of theileriosis have occurred in beef and dairy cattle in subtropical climatic regions (New South Wales) of Australia. There is also one published report of a recent theileriosis outbreak in a beef farm near Seymour in the southeastern state of Victoria. In order to gain an improved insight into the genetic composition of Theileria populations following this outbreak, we undertook herein an integrated PCR-coupled mutation scanning sequencing phylogenetic analysis of sequence variation in part of the major piroplasm surface protein (MPSP) gene within and among samples from cattle involved in the outbreak. Theileria DNA was detected in 89.4% of 94 cattle in the Seymour farm; the genetic analysis showed that the ikeda and chitose genotypes representing the Theileria orientalis complex were detected in 75% and 4.8% of 84 infected cattle, respectively, and that mixed populations of these two genotypes were found in 20.2% of infected cattle. Given unpublished reports of a significant increase in the number of outbreaks in Victoria, future investigations should focus sharply on elucidating the epidemiology of Theileria to subvert the economic impact on the cattle industry in this state. Although used here to explore genetic variation within the T. orientalis complex in Australia, a mutation scanning based approach has broad applicability to other species of Theileria in other countries. Keywords: Cattle / Major piroplasm surface protein (MPSP) gene / Mutation scanning based analysis / Phylogeny / Theileria orientalis DOI 10.1002/elps.201200082 1 Introduction Theileriosis is a tick-borne disease caused by Theileria species, Q1 which are intracellular apicomplexan parasites (piroplasms) that affect a range of wild and domestic animals, particularly ruminants [1]. Depending on the species or subspecies of Theileria, clinical signs can include depression, lymph node swelling, tachypnea, dyspnea, hemolytic anemia, jaundice, abortion, stillbirths and metritis, and mortality is common [1]. Theileria was first detected in cattle in Australia in the 1900s. Theileria infection is thought to have been introduced from Japan into Australia via cattle infested with Hema- physalis longicornis (an ixodid tick) [2] and become endemic Correspondence: Professor Robin B. Gasser, Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia E-mail: robinbg@unimelb.edu.au Fax: + 61-3-9731-2366 Abbreviations: AIC, Akaike information criteria; BI, Bayesian inference; MPSP, major piroplasm surface protein gene; NJ, neighbor-joining; SSCP, single-strand conformation poly- morphism in the northeastern parts of Australia. Traditionally, infection in the cattle in this country was usually asymptomatic and recognized to be linked to Theileria buffeli, now assigned to the Theileria orientalis complex [3, 4], although some clinical cases had been reported in the state of Queensland in the 1960s [2]. Since 2006, there have been theileriosis outbreaks in both beef and dairy cattle, predominantly in the state of New South Wales (NSW) [5]. Because conventional diagnostic methods (based on mor- phology and serology) do not allow the unequivocal identifi- cation of Theileria to species or subspecies, molecular tools have been developed to characterize the piroplasms genet- ically. In particular, DNA sequencing of PCR-amplified re- gions of the major piroplasm surface protein (MPSP) and/or small subunit of nuclear ribosomal RNA (SSU rRNA) genes has allowed the genotypic characterization of isolates [3, 6]. Based on phylogenetic analyses of sequence data for each or both of these loci, members of the T. orientalis complex from cattle can be classified into at least eight genetic groups or ∗ These authors contributed equally to this work. ∗∗ Additional Corresponding Author: Dr. Abdul Jabbar, E-mail: jab- bara@unimelb.edu.au C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com