A second generation multiplex PCR for typing strains of Neospora caninum using six DNA targets.

Genetic diversity of Neospora caninum was investigated through a study of repetitive sequences found in the genome of this species. Twenty different loci were studied, and three were identified that varied in repeat content amongst isolates. No relationship was found between the copy number of repetitive sequences present and host type or geographical location from which the isolates were derived. A multiplex PCR assay was developed for multilocus-strain typing using three microsatellites and three minisatellites, based on the polymorphisms found in the repetitive sequences. This study therefore extends knowledge on the repetitive sequences found in the N. caninum genome and the diversity found within the species. It also provides a second generation multiplex assay that can be used to study the biology of N. caninum. In addition, this study included Neospora hughesi (along with other closely related apicomplexans) as controls. The present study shows N. hughesi to be quite distinct from N. caninum in these repetitive sequences, thereby potentially providing a new approach for the differentiation of these two taxa.