Molecular phylogeny of Pseudocapillaroides xenopi (Moravec et Cosgrov 1982) and development of a quantitative PCR assay for its detection in aquarium sediment.

ABSTRACT

We used high-fidelity PCR to amplify a portion of the small ribosomal subunit (18S rRNA) of Pseudocapillaroides xenopi, a nematode that parasitizes the skin of Xenopus laevis. The 1113-bp amplicon was cloned, sequenced, and aligned with sequences from 22 other nematodes in the order Trichocephalida; Caenorhabditis elegans was used as the outgroup. Maximum-likelihood and Bayesian inference phylogenetic analyses clustered P. xenopi in a clade containing only members of the genus Capillaria. Our analyses support the following taxonomic relationships 1) members of the family Trichuridae form a clade distinct from those in the family Trichocephalida; 2) members of the genera Trichuris and Capillaria form 2 distinct clades within the family Trichuridae; and 3) the genus Trichuris includes 2 distinct clades, one representing parasites that infect herbivores and the other representing parasites that infect omnivores and carnivores. Using 18S rRNA sequence unique to P. xenopi, we developed a Taq Man quantitative PCR assay to detect this P. xenopi sequence in total DNA isolated from aquarium sediment. The assay's lower limit of detection is 3 copies of target sequence in a reaction. The specificity of our assay was validated by using negative control DNA from 9 other pathogens of Xenopus. Our quantitative PCR assay detected P. xenopi DNA in the sediment of 2 of 12 aquaria from the source institution of the specimen used to develop the assay; these aquaria had been treated with ivermectin 6 mo previously.