Determination of Trichuris muris from murid hosts and T. arvicolae (Nematoda) from arvicolid rodents by amplification and sequentiation of the ITS1–5.8S-ITS2 segment of the ribosomal DNA

Abstract

Trichuris muris has been isolated from murid hosts ( Apodemus sylvaticus and Mus musculus) and Trichuris arvicolae from arvicolid rodents in Barcelona, Spain. Genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The ITS2 of both populations isolated from Apodemus and Mus was 382 nucleotides in length and had a GC content of about 60.73%, while the ITS2 of T. arvicolae was 442 nucleotides in length and had a GC content of about 59.8%. Furthermore, the ITS1 of Trichuris from murids was 448 nucleotides in length and had a GC content of about 56.47%, while T. arvicolae was 446 nucleotides in length and had 57.62% of GC content. A total of 161 and 173 nucleotides were observed along the 5.8S gene of T. murisand T. arvicolae, respectively; This difference in nucleotides was due to the insertion of a DNA segment (transposon) in the 5.8S sequence of the latter species. Slight intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all of the individuals assayed. Sequence analysis of the internal transcribed spacers and the 5.8S gene demonstrated no sequence differences between T. muris isolated from both of its murid hosts. Nevertheless, clear differences were detected between the ITS2, ITS1 and 5.8S gene of T. muris and T. arvicolae. This corroborates the existence of two separate Trichuris species in murid and arvicolid hosts. Furthermore, a phylogenetic analysis was carried out and endonucleases restriction maps were elaborated for both species.

Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts

“…Furthermore, intraspecific variation was observed in the ITS1 of T. vulpis (1.22%), of T. suis from wild boar (0.45%), and in the ITS2 of T. vulpis (1.6%). These variations observed within species in T. vulpis were higher than those obtained for the ITS2 and ITS1 of T. muris (1.04 and 0.44%, respectively) and T. arvicolae (0.7 and 0%, respectively) (Cutillas et al 2002). In nematodes, multiple different sequences of the rDNA ITS are frequently found within individual worms (Back et al 1984;Anderson 1995;Hoste et al 1995;Brattey and Davidson 1996).…”

“…The presence of nucleotide repeats was also reported in the ITS1 fragment of trichurids from murids (Cutillas et al 2002) in the ITS2 of T. ovis and T. leporis (Oliveros et al 2000) and in the ITS1 sequences of T. skrjabini, T. leporis, T. ovis, and T. globulosa (Cutillas et al 2004). Sequences of this nature generally occur as randomly dispersed repetitive elements in virtually all eukaryotic genomes where their localization within the genome appears to be relatively stable (Moore et al 1991;Schlötterer et al 1991).…”

“…These intraindividual variations (polymorphism) were detected in the ITS1 of T. skrjabini, T. ovis, Trichuris globulosa, and T. leporis and in the ITS2 of T. skrjabini (Cutillas et al 2004). Furthermore, this intraindividual variation was cited in the ITS1 and ITS2 of T. muris and T. arvicolae (Cutillas et al 2002). The existence of polymorphism was cited in the ITS2 of T. ovis and T. leporis (Oliveros et al 2000) 1998), in the ITS+ sequences of strongylid nematodes (Campbell et al 1995;Hoste et al 1995;Stevenson et al 1995), and Echinococcus granulosus (Bowles and McManus 1993).…”

“…Sequence alignments were obtained using the PileUp program of the Wiscosin Genetics Computer Group (GCG) package. Furthermore, data of the ITS1-5.8S-ITS2 from other species of trichurids and ITS2 sequence from T. suis from S. scrofa domestica (swine) (Cutillas et al 2002;Oliveros et al 2000;Cutillas et al 2004) were used for comparison.…”

Cytochrome oxidase subunit 1 and mitochondrial 16S rDNA sequences of Trichuris skrjabini (Tricocephalida: Trichuridae)

“…Nevertheless, the sequences obtained from ribosomal internal transcribed spacer (ITS) regions and 5.8S gene of different species of Trichuris: T. ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris suis (Oliveros et al 2000;Cutillas et al 2002Cutillas et al , 2007 could not be used for phylogenetic analyses. Since mitochondrial genome sequences have provided useful markers for systematics and phylogenetics of organisms (Avise 1994;Hu and Gasser 2006), we chose to determine the partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox 1) and partial mitochondrial 16S ribosomal DNA sequence of T. skrjabini.…”