ITS-2 rDNA Sequencing of Gnathostoma Species (Nematoda) and Elucidation of the Species Causing Human Gnathostomiasis in the Americas

Abstract

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.

Emerging helminth zoonoses

“…Although previous reports had identi®ed G. spinigerum as the aetiologic agent of gnathostomiasis in Mexico, recent evidence suggests that cases identi®ed as being due to G. spinigerum are, in fact, due to Gnathostoma binucleatum [50]. At least two other Gnathostoma species appear to be unique to Mexico and have been implicated in human infection [50].…”

“…At least two other Gnathostoma species appear to be unique to Mexico and have been implicated in human infection [50].…”

Proteases secreted by Gnathostoma binucleatum degrade fibronectin and antibodies from mammals

“…The alignment of the 421 bp product of ITS-2 showed 100 % identity with the sequences reported for G. binucleatum (Almeyda-Artigas et al, 2000;León-Regagnon et al, 2002) and 99.76% with the nucleotide sequence of a parasite of the same species obtained by biopsy (Almeyda-Artigas et al, 2000), indicating that the larvae used to obtain the ESP were indeed G. binucleatum. The ESP showed proteolytic activity against bovine gelatin at both pH 8 and pH 4.5.…”

“…The bands with protease activity at 80 and 56 kDa are able to hydrolyze proteins from the extracellular matrix, suggesting that they could be involved in penetrating or migrating through tissues, as has been described for other helminthes (Berasain et al, 1997;Knox, 2011). The infective larvae of the nematode Ancylostoma caninum secrete proteases involved in migration through host tissues (Williamson et al, 2011); moreover, proteolytic activity by a protease against collagen and fibrinogen has been reported in the ESP of third-stage larvae of the nematode Lagochilascaris minor (Barbosa et al, 2006). In addition, the bands with protease activity at 80 and 56 kDa were able to degrade both sheep and human gamma globulins.…”

Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum

“…Primers which amplify the ITS-2 region of snails (Almeyda-Artigas et al, 2000;Caron et al, 2011) and the second expansion segment of 28S rRNA in ants (Belshaw and Quicke, 1997) were used to identify the snails and ants to species level, and to ensure DNA extraction and PCR amplification were successful. Table 1.…”