Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA

Abstract

Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecum osculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.

Import of exotic and zoonotic trematodes (Heterophyidae: Centrocestus sp.) in Xiphophorus maculatus: Implications for ornamental fish import control in Europe

“…The reaction mixtures consisted of 6 µl lysate as template, 1 unit of BioTaq DNA polymerase (DNA-Technology), 1 mM dNTP, 1.5 mM MgCl 2 and 1 µM of the two primers. In order to amplify the ITS region, the primers NC5 (5'-GTA GGT GAA CCT GCG GAA GGA TCA TT-3') and NC2 (5'-TTA GTT TCT TTT CCT CCG CT-3') were used as forward and reverse primer, respectively (Zhu et al 2007). The PCR procedure consisted of a pre-denaturation step at 95°C for 2 minutes, followed by 45 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and elongation at 72°C for 2 minutes, and finally post-elongation at 72°C for 5 minutes.…”

Morphological and molecular characterization of Raphidascaris (Ichthyascaris) lophii (Wu, 1949) (Nematoda, Anisakidae) from marine fishes from China, with a key to the species of the subgenus Ichthyascaris

“…Recently, the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) has been successfully employed as a molecular marker for the accurate identification of ascaridoid nematodes (Zhang et al 2007;Zhu et al 2007;Du et al 2010;Fang et al 2010;Testini et al 2011;Li et al 2012). Thus, the …”

Host size-dependent anisakid infection in Baltic cod Gadus morhua associated with differential food preferences

“…The reaction mixtures consisted of 6 µl lysate as template, 1 unit of BioTaq DNA polymerase (DNA-Technology), 1 mM dNTP, 1.5 mM MgCl 2 and 1 µM of the 2 primers. In order to amplify the internal transcribed spacer (ITS) region, the primers NC5 (5'-GTA GGT GAA CCT GCG GAA GGA TCA TT-3') and NC2 (5'-TTA GTT TCT TTT CCT CCG CT-3') were used as forward and reverse primer, respectively (Zhu et al 2007). PCR conditions were 2 min of pre-denaturation at 94°C followed by 36 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s and elongation at 72°C for 1 min 15 s. Finally, a post-elongation step was performed at 72°C for 7 min.…”