Opisthorchis viverrini: Identification of a glycine–tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis q Jiraporn Ruangsittichai a , Vithoon Viyanant b , Suksiri Vichasri-Grams a , Prasert Sobhon c , Smarn Tesana d , Edward Suchart Upatham e , Annemarie Hofmann f , Gu ¨ nter Korge f , Rudi Grams b, * a Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand b Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand c Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand d Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand e Faculty of Science, Burapha University, Chonburi, Thailand f Institut fu ¨ r Biologie, Genetik, Freie Universita ¨ t Berlin, Arnimallee 7, D-14195 Berlin, Germany Received 10 April 2006; received in revised form 14 June 2006; accepted 16 June 2006 Abstract A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite’s excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82–0.48) and specificity (0.97– 0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Eggshell protein; Expression; Localization; Molecular cloning; Opisthorchis viverrini; Serodiagnosis; Trematode 1. Introduction Opisthorchiasis, caused by the human liver fluke Opisthorchis viverrini, is an important food-borne parasitic disease in many parts of Southeast Asia including Thai- land, Lao PDR, Vietnam, and Cambodia (Sithithaworn and Haswell-Elkins, 2003; WHO, 2004). Recently, it has been estimated that about six to eight million people are infected with the parasite in Thailand (Jongsuksuntigul et al., 2001; Jongsuksuntigul and Imsomboon, 2003; WHO, 2004). Infected people in underdeveloped areas without proper sanitation can contribute to the spread of the parasite by passing its eggs with feces into water reser- voirs. Opisthorchiasis is characterized by hepatobiliary diseases, including cholangitis, obstructive jaundice, hepa- tomegaly, cholecystitis, and cholelithiasis (see review in Sripa, 2003). In chronic infections with high-worm bur- dens, opisthorchiasis is associated with the formation of 0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.06.012 q Nucleotide sequences data reported in this paper are available in the EMBL, GenBank, and DDJB databases under the Accession No. DQ479316. * Corresponding author. Tel.: +66 2 9869213x7074; fax: +66 2 5165379. E-mail address: rgrams@alpha.tu.ac.th (R. Grams). www.elsevier.com/locate/ijpara International Journal for Parasitology 36 (2006) 1329–1339