Invited review
Mutation scanning-coupled tools for the analysis of genetic variation in Taenia and diagnosis – Status and prospects

https://doi.org/10.1016/j.meegid.2009.05.005Get rights and content

Abstract

Cestodes of the genus Taenia occur as adult tapeworms in the small intestine of carnivorous definitive hosts and are transmitted to particular mammalian intermediate hosts, in which they develop as fluid-filled larvae in tissues, causing the disease cysticercosis or coenuriasis. A number of species are of medical importance and/or cause losses to the meat and livestock industry mainly due to the condemnation of infected muscle and offal. The control of taeniid cestodes relies upon epidemiological data, including the precise identification and characterization of the causative agents. Traditional, phenetic techniques have limitations for specific diagnosis. Although there has been progress in the establishment of molecular tools, there has been relatively limited application of mutation scanning approaches to species of Taenia. In the present article, we briefly review key genetic markers used for the specific identification of taeniids and tools for the analysis of genetic variation within and among populations and the diagnosis of taeniasis and cysticercosis/coenuriasis. We also discuss the advantages and disadvantages of selected techniques and emphasize the benefits of utilizing mutation scanning-based approaches in achieving detailed insights into the population genetics and epidemiology of Taenia species.

Introduction

Adult tapeworms of the genus Taenia (family Taeniidae = taeniids) infect the small intestines of carnivorous definitive hosts and are transmitted (via eggs) to mammalian intermediate hosts in which they become established as larval stages (= cysts) in specific tissues, causing the disease cysticercosis or coenuriasis (Bowman, 2008). The larval (including cysticerus and coenurus) stages of a number of Taenia species cause losses to the meat industry due to the condemnation of infected meat or offal, and/or are zoonotic (Jones and Pybus, 2001, Ito et al., 2003a, Carabin et al., 2005, Flisser et al., 2006, Schantz, 2006).

The specific diagnosis of Taenia infections in both definitive and intermediate hosts is central to the epidemiology and control of cysticercosis and coenuriasis. Traditionally, the specific identification of Taenia species has been based predominantly on ecological, biological and/or morphological criteria, including the features of the adult stage (such as the number, size and shape of the rostellar hooks, the presence or absence of a vaginal sphincter, the location of the genital pore along the segment margin and the number of principal lateral branches of the gravid uterus, the distribution of the testes, and the shape of the cirrus-sac and its extent relative to the longitudinal osmoregulatory canals), the morphology and type of asexual reproduction of the larval stage, and the level of host specificity in different geographical regions (Verster, 1969, Beveridge and Gregory, 1976, Edwards and Herbert, 1981, Rausch, 1994, Loos-Frank, 2000, Chervy, 2002, Rausch, 2003). However, based on these criteria, unequivocal identification is often difficult.

Biochemical and traditional molecular approaches, such as multilocus enzyme electrophoresis (MEE) and Southern blot-coupled restriction fragment length polymorphism (RFLP) analysis, have assisted in the genetic characterization and identification of Taenia spp. from different hosts (reviewed by McManus, 1990a, McManus, 1990b, McManus and Bowles, 1996). Techniques based on the use of the polymerase chain reaction (PCR; Saiki et al., 1988) have found broader applicability to epidemiological and/or population genetic studies of some taeniid cestodes, particularly Echinococcus, mainly because their sensitivity permits the analysis of particular genes from tiny amounts of genomic DNA from fresh, frozen or even ethanol fixed parasite material (see McManus, 2006, Yamasaki et al., 2006a). Nonetheless, there has been limited molecular study of the broad range of species of Taenia recorded to date (Verster, 1969, Loos-Frank, 2000). In the present article, we provide an account of genetic markers employed for the identification of Taenia species, and tools for the analysis of genetic variation within and among species and the diagnosis of cysticercosis/coenuriasis and/or taeniasis. We also discuss the advantages and disadvantages of selected techniques and emphasize the benefits of utilizing “analytical” and “diagnostic” mutation scanning to achieve better insights into the systematics, epidemiology and population genetics of members of the genus Taenia.

Section snippets

Key genetic markers and methods used, and their attributes

PCR-coupled techniques, employing specific primer pairs/sets for the selective amplification of different genetic loci, followed by enzymatic cleavage, or sequencing, have been used often to identify, characterize or classify Taenia species or “genotypes” (Campbell et al., 2006, McManus, 2006, Varcasia et al., 2006, Yamasaki et al., 2006a). The key loci used are within mitochondrial (mt) genes or nuclear ribosomal DNA.

Mitochondrial DNA (mtDNA) has been used most commonly for the identification

Mutation scanning-coupled tools for the assignment of Taenia to species or genotypes (strains or subspecies), and systematic and population genetic studies

Base excision sequence scanning (BESS; Hawkins and Hoffman, 1997) is a PCR-based mutation scanning method that detects and/or locates DNA mutations. The BESS method includes two procedures that generate “T” (BESS T-Scan) and “G” ladders (BESS G-Tracker), analogous to the T and G ladders of conventional dideoxy sequencing, respectively. According to Hawkins and Hoffman (1997), the procedures are relatively simple to carry out and do not require specialized equipment or optimization beyond PCR.

Protocol for integrated SSCP-based analysis

Based on recent published (Zhang et al., 2007) and unpublished evaluations, the following SSCP-phylogenetic approach is now applied to taeniids in our laboratory:

  • 1.

    Extract total genomic DNA from individual isolates (preferably individual adults or larvae) using a small-scale proteinase K digestion procedure. Add ∼50–100 μl packed volume of parasite material to a tube with 100–200 μl of H2O containing 1% (w/v) sodium dodecyl-sulphate and incubate at 37 °C for 12–14 h. Vortex tube, centrifuge at 12,000 ×

Prospects for semi-automated mutation scanning tools

There have been significant enhancements in automated electrophoretic platforms, due to an increased demand for high throughput genetic analysis of pathogens and in silico-handling of data. This comment applies, for instance, to the different capillary electrophoretic (CE) systems (reviewed by Righetti et al., 2002, Mitchelson, 2003). CE-coupled SSCP has been employed as a high-throughput method for a range of applications, including the detection of SNPs (Doi et al., 2004, King et al., 2005,

Concluding remarks

In our hands, PCR-coupled SSCP has proved to be practical, inexpensive, sensitive, specific and reproducible. Provided that it is applied according to a standardized protocol (thus achieving a high mutation detection rate) and that results are interpreted carefully, SSCP has major analytical capacity and, together with selective sequencing and phylogenetic analyses of sequence data, is well suited to studies of the systematics, population genetics and epidemiology of taeniids, provided careful

Acknowledgements

Thanks to colleagues, including Ian Beveridge, Neil Chilton, Dennis Jacobs and Lynda Gibbons, for cooperation and collaborations. Our genetic studies of taeniids were supported by a small grant from the Australian Research Council (ARC) in 1994. RBG's laboratory is currently in receipt of grants from the Australian Research Council (ARC), Melbourne Water Corporation, and Meat and Livestock Australia (MLA); support from the American-Australian Fulbright Commission is gratefully acknowledged.

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