Characterization of Toxascaris leonina and Tococara canis from cougar (Panthera leo) and common wolf (Canis lupus) by nuclear ribosomal DNA sequences of internal transcribed spacerss

In the present study, samples representing Toxascaris leonina and Tococara canis were collected from cougar (Panthera leo) and common wolf (Canis lupus) from Harbin Northern Forest Zoo in Heilongjiang Province, China, and they were characterized by nuclear ribosomal DNA (rDNA) sequences of internal transcribed spacers (ITS). The ITS rDNA was amplified by polymerase chain reaction (PCR), then sequenced and compared with that of other members of ascaridida published in GenBank (TM). Furthermore, the phylogenetic relationship was reconstructed using Maximum Parsimony and Bayesian analyses. For the T. leonina of cougar, the length of the ITS sequences was 698 bp (the ITS1 and ITS2 were 419 and 279 bp, respectively) and the corresponding sequences of T. canis from common wolf were 808 bp (the ITS1 and ITS2 were 485 and 323 bp, respectively). The identity of ITS sequences of T. leonina and T. canis from cougar and common wolf was 76%. The ITS sequences were analyzed by PCR-linked restriction fragment length polymorphism (PCR-RFLP) which was established for the unequivocal delineation of the T. leonina and T. canis with which were easily confused morphologically, using restriction endonuclease EcoRV and SalI. The valuable tool established in this study makes the molecular identification, the ecology and genetic structure studies of these two ascaridoids possible.