D. C. Guevara

Trichuris suis and Trichuris trichiura are different nematode species

In this paper, a morphological and biometrical study by optical microscopy and scanning electronic microscopy (SEM) of Trichuris suis isolated from different hosts (Sus scrofa domestica and Sus scrofa scrofa) and Trichuris trichiura isolated from chimpanzee, has been carried out. Our results demonstrate the existence of typical pericloacal papillae in both species. Biometrical parameters of T. suis and T. trichiura overlapped but males and females of T. trichiura tended to be shorter and thinner than those of T. suis. Our results suggest that T. suis and T. trichiura cannot be differentiated using standard procedures as morphological and biometrical determinations. Thus, the ITS1-5.8S-ITS2 region of the ribosomal DNA was sequenced to allow a differentiation between T. suis and T. trichiura on genetic level. The ITS1 and ITS2 sequences derived from T. trichiura eggs isolated from feces of primates (Colobus guereza kikuyensis and Nomascus gabriellae) showed clear differences to the respective sequences of T. suis derived from eggs of different porcine hosts. The 5.8S gene was similar between the two species. Sequences obtained from different populations of the same species showed no significant differences indicating that the ITS1-5.8S-ITS2 sequences reported in this study are representative for T. trichiura and T. suis, respectively. Phylogenetic relationships have been determined attending to the ITS1 and ITS2 sequences from different species of the genus Trichuris. In conclusion, T. trichiura and T. suis are considered to be closely related but genetically different species. Both species can be easily and reliably distinguished by a PCR-RFLP analysis of the ITS1 and ITS2 sequences with different restriction enzymes.

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. muris and 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.

Determination of Trichuris skrjabini by Sequencing of the ITS1–5.8S–ITS2 Segment of the Ribosomal DNA: Comparative Molecular Study of Different Species of Trichurids

Adults of Trichuris skrjabini have been isolated from the cecum of caprine hosts (Capra hircus), Trichuris ovis and Trichuris globulosa from Ovis aries (sheep) and C. hircus (goats), and Trichuris leporis from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and the ITS1–5.8S–ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced by polymerase chain reaction (PCR) techniques. The ITS1 of T. skrjabini, T. ovis, T. globulosa, and T. leporis was 495, 757, 757, and 536 nucleotides in length, respectively, and had G + C contents of 59.6, 58.7, 58.7, and 60.8%, respectively. Intraindividual variation was detected in the ITS1 sequences of the 4 species. Furthermore, the 5.8S sequences of T. skrjabini, T. ovis, T. globulosa, and T. leporis were compared. A total of 157, 152, 153, and 157 nucleotides in length was observed in the 5.8S sequences of these 4 species, respectively. There were no sequence differences of ITS1 and 5.8S products between T. ovis and T. globulosa. Nevertheless, clear differences were detected between the ITS1 sequences of T. skrjabini, T. ovis, T. leporis, Trichuris muris, and T. arvicolae. The ITS2 fragment from the rDNA of T. skrjabini was sequenced. A comparative study of the ITS2 sequence of T. skrjabini with the previously published ITS2 sequence data of T. ovis, T. leporis, T. muris, and T. arvicolae suggested that the combined use of sequence data from both spacers would be useful in the molecular characterization of trichurid parasites.

Phylogenetic Relationships in Rhinonyssid Mites (Acari: Rhinonyssidae) Based on Mitochondrial 16S rDNA Sequences

A 390 bp region of the 16S rDNA gene was sequenced from six species ofrhinonyssid mites (Tinaminyssus columbae, T. minisetosum, Sternostomaturdi, S. sternahirundo, S. fulicae and Ptilonyssus euroturdi) andtwo subspecies (Tinaminyssus melloi melloi andTinaminyssus melloi streptopeliae) to examine the level ofsequence variation and the taxonomic levels to show utility in phylogenyestimation. Furthemore, two different geographic locations of T. m.melloi and T. m. streptopeliae were analyzed todetect variation between populations. Molecular data revealed the existence oftwo distinct groups in the genus Tinaminyssus parasitic oncolumbiform birds. These results are in agreement with those reported by someauthors using morphological characters. Sternostoma turdi parasitizing aerial birds appeared to be phylogenetically separated from otherspecies of this genus isolated from aquatic birds. Moreover, our studyaddressesthe validity of the subspecies status of T. melloistreptopeliae. This region of the mitochondrial 16S rDNA gene is auseful marker for inferring phylogenetic relationships among closely relatedrhinonyssid species, but not for more distantly related taxa.

Molecular study on three morphotypes of Demodex mites (Acarina: Demodicidae) from dogs

Canine demodicosis is a severe and highly prevalent dermatologic disease in dogs. Pet dogs can be affected by three recognized Demodex species that can produce clinical effects. In this paper, three morphological types of Demodex mites have been isolated from Spanish dogs. A complete morphobiometrical study of each one has been carried out. Morphological and biometrical studies revealed three closely related populations with some distinctive characteristics and could be identified as Demodex canis, Demodex injai, and Demodex sp. “cornei.” Furthermore, one population of D. canis from China, different populations of Demodex folliculorum from human skin (Spain and China), D. folliculorum from human eyelashes (Spain), and Demodex brevis from human skin (China) were considered to find out the level of variation between different species and geographical origin. The aim of the present study is to assess the usefulness of mitochondrial DNA molecular markers in establishing phylogenetic relationships and resolve taxonomic questions in Demodex mites. Molecular studies based on the amplification and sequencing of the 16S rDNA and cytochrome oxidase I mitochondrial genes did not show clear differences between the three morphotypes considered. Furthermore, phylogenetic relationships in Demodex mites were analyzed. The resulting phylogenetic trees show that Demodex species from dogs were gathered together, and populations of D. folliculorum from humans appear together in a different branch; however, D. brevis from humans seemed to be more distant. Our results show that cytochrome oxidase I region is a useful tool to solve different taxonomic questions at the species and population level and to infer phylogenetic relationships in Demodex species. However, 16S mitochondrial rDNA seems a good marker for comparisons at an interspecies level, but not at a population level in this group of mites. Furthermore, from genetic distance and divergence data, we would suggest that D. canis, D. injai, and Demodex sp. cornei are polymorphisms of the same species.

Morphobiometrical and molecular study of two populations of Demodex folliculorum from humans.

A morphobiometrical and molecular study of two populations of Demodex folliculorum from humans isolated from different habitats, skin and eyelashes follicles, were carried out. Morphological and biometrical studies revealed two closely related populations with any distinctive characteristics. For molecular study, a 436-bp region of the 16S rDNA gene and a 453-bp region of the cytochrome oxidase I (COI) gene from individual mites of each population considered were sequenced. Intraindividual and interindividual sequence variation was studied in both populations. Our data show that 16S rDNA is not a useful marker to discriminate between populations; however, COI gene sequences can help to identify the two populations considered, which are morphologically very close and difficult to separate by classic methods. These results are in agreement with the morphological and biometrical differences detected between D. folliculorum from eyelashes and human skin. This study appeals for the revision of the taxonomic status of the D. folliculorum populations, as well as for the species included within genus Demodex.

16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis).

Utility of ITS1-5.8S-ITS2 and 16S mitochondrial DNA sequences for species identification and phylogenetic inference within the Rhinonyssus coniventris species complex (Acari: Rhinonyssidae)

The complete internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2 region of the ribosomal DNA and a 390-bp region of the 16S rDNA gene from five taxa belonging to Rhinonyssus (Rhinonyssus vanellus, Rhinonyssus tringae, Rhinonyssus neglectus, Rhinonyssus echinipes from Kentish plover, and Rhinonyssus echinipes from grey plover) were sequenced to examine the level of sequence variation and the taxonomic levels to show utility in phylogeny estimation. Our data show that these molecular markers can help to discriminate between species and populations included in the Rhinonyssus coniventris complex (R. tringae, R. neglectus, R. echinipes), which are morphologically very close and difficult to separate by classic methods. A comparative study with sequences from other rhinonyssid mites previously published was also carried out. The resulting phylogenetic tree inferred from ITS1–5.8S–ITS2 region sequences obtained in this paper, together with those from other 11 taxa of rhinonyssid, shows slight differences from the current taxonomy of the Rhinonyssidae. This study appeals for the revision of the taxonomic status of the R. coniventris complex, as well as for the species included within it.

Characterization of Trichuris skrjabini by isoenzyme gel electrophoresis: comparative study with Trichuris ovis

Morphological and biometric studies were performed in Trichuris skrjabini (Baskakov, 1924) collected from the caecum of Capra hircus. The LDH (EC 1.1.1.27.), G6PD (EC 1.1.1.49.), GPI (EC 5.3.1.9.), MDH (EC 1.1.1.37) and malic enzyme (ME) (EC 1.1.1.40) isoenzymatic patterns of T. skrjabini were determined by starch gel electrophoresis. The G6PD and GPI isoenzymatic patterns of T. skrjabini displayed two anodic bands for both enzymes: one fast migration band and one band near the origin. This isoenzymatic pattern was interpreted as two gene loci encoding both enzymes. The LDH isoenzymatic pattern of T. skrjabini was characterized by the presence of a cathodically migrating band, while the MDH isoenzymatic pattern showed a very slow cathodic band. These two phenotypes were interpreted as the expression of a homozygous state of a gene locus for LDH and MDH in T. skrjabini. The ME isoenzymatic pattern was characterized by the presence of a single anodic band. Further, comparative isoenzymatic studies were carried out between T. skrjabini and T. ovis. The different G6PD, GPI, LDH, MDH and ME isoenzymatic patterns observed for both species allowed us to distinguish them and therefore to use isoenzymatic patterns as a diagnostic tool to differentiate species of Trichuris.

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

The partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox 1) and partial mitochondrial 16S ribosomal DNA of Trichuris skrjabini (Baskakov 1924) isolated from Capra hircus have been amplified and sequenced. The analyses of multiple sequence alignments of mitochondrial 16S rDNA and cox 1 of T. skrjabini revealed high homology with those of Trichinella species. For the first time, the mitochondrial DNA gene sequences of one species of trichurid nematode have been cited.