Research Article Mutation scanning-based analysis of anisakid larvae from Sillago flindersi from Bass Strait, Australia Anisakidosis is an important fish-borne disease caused by the larvae of anisakid nema- todes, which affects humans and a range of other animals. The accurate identification of members of this nematode group is central to investigating the epidemiology of the parasites and in the surveillance and control of anisakidosis. It is now well known that morphological identification alone does not allow specific identification, particularly of larval stages. To better understand the epidemiology of anisakid nematodes in southern Australian fishes and the potential risks posed to human health, a survey of 50 speci- mens of the commercially important fish, Sillago flindersi, from Bass Strait, Australia was conducted. We characterised anisakid larvae by PCR-coupled mutation scanning, sequencing and phylogenetic analyses of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. This study revealed that 92% of the S. flindersi examined were infected with anisakids (n 5 194), which were represented by seven genotypes. Phylogenetic analyses of the genotypes defined herein, together with reference sequence for Anisakis pegreffii and Hysterothylacium sp. from public databases (i.e. GenBank), revealed the presence of A. pegreffii (n 5 24), Hysterothylacium larval type IV (n 5 90) and Hysterothylacium larval type VIII (n 5 80) in S. flindersi. Thus, the PCR- coupled mutation scanning approach employed herein is an effective tool for the genetic characterisation of anisakid nematodes for diagnostic and analytical purposes (nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN631796-809). Keywords: Anisakidae / Internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) / Nematoda / Single-strand conformation polymorphism DOI 10.1002/elps.201100438 1 Introduction Anisakidosis is a parasitic disease caused by larval anisakid nematodes [1]. In humans, this disease has been reported from five continents, including Asia (endemic foci mainly in south-east Asia), Africa, the Americas, Australia and Europe [2, 3]. Although members of the genus Anisakis are considered as the most common cause of anisakidosis [2, 4], species of Pseudoterranova, Contracaecum and Hyster- othylacium have also been reported to infect humans [5–7]. The life cycle of anisakids is complex. The adult nematodes live in the stomach of marine mammals (i.e. whales, dolphins, seals, sea lions and/or walruses), fish-eating birds and large pelagic teleosts, eggs are expelled with the faeces, embryonate and then hatch to release free-living, infective third-stage larvae (L3). These L3s are ingested by euphau- siids or copepods, which then are ingested by fish or cephalopods (paratenic hosts), in which the L3s are embedded in the viscera and muscles or remain free in the body cavity. Definitive hosts (marine mammals, birds or teleosts) become infected when they ingest either infected intermediate or paratenic hosts containing L3s, in which two moults occur, before they develop to dioecious adults and produce eggs [2, 3]. Anisakidosis occurs when people accidentally ingest L3s in infected raw, undercooked or improperly processed fish or squid. Usually within a few hours of ingestion of infective larvae, patients may suffer from violent abdominal pain, nausea, vomiting, and/or diarrhoea [1], some patients developing syndromes asso- ciated with variable clinical signs [8], which may include isolated cutaneous swellings, urticaria and even life-threa- tening anaphylactic shock to chronic, debilitating conditions Abdul Jabbar 1 Avalene T. W. Khoon 1 Teo X. Hui 1 Bjoern C. Schaeffner 1 Aaron R. Jex 1 Matthew J. Nolan 1 Andreas Lopata 2 Robin B. Gasser 1 Ian Beveridge 1 1 Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia 2 Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland, Australia Received August 8, 2011 Revised August 31, 2011 Accepted September 12, 2011 Abbreviations: AIC, Akaike Information Criteria; BI, Bayesian inference; ITS, internal transcribed spacer; NJ, Neighbour- joining; SSCP, single-strand conformation polymorphism Correspondence: Dr. Abdul Jabbar, Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia E-mail: jabbara@unimelb.edu.au Fax: 161-3-9731-2366 & 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2012, 33, 499–505 499