RESEARCH BRIEF Taenia ovis: Copy Number of Genes Encoding Host-Protective Antigens Determined by Competitive PCR Jacqueline G. Waterkeyn,* ,1 Alan F. Cowman,† and Marshall W. Lightowlers* *Veterinary Clinical Centre, The University of Melbourne, Princes Highway, Werribee, Victoria 3030 Australia; and †The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050 Australia WATERKEYN, J. G., COWMAN, A. F., AND LIGHTOWLERS, M. W. 1997. Taenia ovis: Copy number of genes encoding host-protective antigens determined by competitive PCR. Experimental Parasitology 86, 84–88. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: cysticercosis; competitive PCR; gene copy number; vaccine antigens; Cestoda. Antigenic diversity and gene copy number have impor- tant implications for the use of defined antigen vaccines for control of parasitic infections, especially over a wide geo- graphic area. The 45W-based vaccine against the cestode parasite of sheep, Taenia ovis, provides a valuable model for study of gene polymorphism and antigenic diversity in a host-protective vaccine antigen. Johnson et al. (1989) de- scribed the isolation of a cDNA designated 45W, which expressed a protein capable of inducing protective immune responses in vaccinated sheep. The gene encoding 45W is a member of a small gene family (Waterkeyn et al. 1995). All members of the family are expressed by T. ovis including one, designated 45S, which also encodes a host-protective antigen (Waterkeyn et al., in press; Lightowlers et al. 1996). Differential expression of gene family members can, under appropriate selection conditions, provide a means for host immune evasion, as occurs in some protozoan parasites such as the trypanosome variable surface glycoprotein and ma- laria var genes (Borst et al. 1995). Moreover, differences in gene copy number may contribute to phenotypic alterations in protozoan parasites (Cowman et al. 1994). Polymor- phisms in gene sequences and gene copy number have not been reported for a host-protective antigen in the parasitic metazoa. Preliminary investigation of T. ovis genomic DNA by Southern blot using 45W cDNA as a probe revealed varia- tion between different T. ovis isolates. However, hybridiza- tion could not be used to assess gene copy number differ- ences in the 45W multigene family. Genomic DNA frag- ments of the 45W gene family vary significantly in their signal strength using 45W as a probe under conditions of high stringency (Waterkeyn et al. 1995). Hence, related genes present in the T. ovis genome may be masked by superimposable fragments of a higher signal strength, giv- ing a false quantitative assessment. In addition, the broad range of intensities of the various fragments does not allow a linear densitometry-based approach to be used in assess- ment of gene copy number since intensities in autoradio- graphs do not lie within the linear range of quantitation using a single exposure of the Southern blot membrane. In this report, we outline a quantitative approach based on competitive ‘‘hot-start’’ PCR (Siebert and Larrick 1992; Kellogg et al. 1994) to assess the number of 45W and 45S genes, encoding host-protective antigens in individual T. ovis isolates. Total genomic DNA was extracted from adult T. ovis tapeworms of Australian or New Zealand origin by the method described by Waterkeyn et al. (1995). Tapeworms were positively identified as T. ovis on morphological cri- teria (Beveridge and Gregory 1976) as well as their ability to infect sheep with typical cysticerci of T. ovis. DNA was digested with EcoR1 endonuclease (Pharmacia, Uppsala Sweden) and electrophoretically separated on 0.6% agarose gels. Digestion products were transferred to nylon filters by capillary action (Southern 1975) and hybridized to digoxi- genin-labeled 45W cDNA as previously described (Water- keyn et al. 1995). Worms were found to conform to one of two distinct restriction patterns, designated alpha () and beta () (Fig. 1). The -parasites could be distinguished from  by virtue of an extra band of high signal strength (III, 10.5 kb) between fragments II and III in Southern experiments and an increase in signal strength of band VII (Fig. 1a). To exclude the possibility that the observed varia- tion was due to either partial digestion or an EcoR1 poly- morphism, duplicate samples were hybridized to DIG- labeled T. ovis dynamin, which is known to exist as a single copy in the T. ovis genome (Fig. 1b, unpublished observa- tions). This confirmed that (i) the DNA had been completely cut and (ii) equivalent amounts of DNA had been loaded per well. Analysis using a panel of restriction enzymes also 1 To whom correspondence should be addressed at The Walter and Eliza Hall Institute of Medical Research, Park- ville, Victoria 3050, Australia. Fax: (61-3) 9347-0852. E- mail: waterkeyn@wehi.edu.au. EXPERIMENTAL PARASITOLOGY 86, 84–88 (1997) ARTICLE NO. PR974157 84 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.