Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences.

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.

Cloning and sequence analysis of the DNA polymerase alpha gene of Leishmania donovani: comparison with the human homologue.

The gene encoding the DNA polymerase alpha catalytic subunit of the kinetoplastid parasite L. donovani has been isolated, sequenced and compared with other eukaryotic homologues. The coding region is 4020 bp in length and specifies an inferred protein sequence of 1339 amino acids (aa). There is a high level of variability between the human and L. donovani gene sequences, but functional substrate-binding residues identified in humans and yeast appear to also be conserved in this parasite. The discovery of a cysteine-rich region located in the midst of the active sites of the enzyme, which appears to be unique to the Kinetoplastids, and aa differences found between some of the conserved regions implicated in catalytic function, may aid in drug design. The putative DNA binding Zn finger at the C-terminus of the protein appears highly species specific and may have potential as a drug target for blocking enzyme catalysis in the parasite.

Phylogenetic relationships between Toxoplasma and Sarcocystis deduced from a comparison of 18S rDNA sequences.

The current taxonomy of parasites in the genus Sarcocystis is largely based on morphological characteristics as well as on host specificity and life-cycle structure. Recently, phylogenetic analyses of partial ribosomal RNA (rRNA) sequences provided support for paraphyly of Sarcocystis. We have tested the validity of this hypothesis by sequencing the complete 18S rRNA genes of Sarcocystis arieticanis, Sarcocystis gigantea and Sarcocystis tenella and comparing them with gene sequences derived from other taxa of the phylum Apicomplexa. The results obtained from this study do not reject the hypothesis of monophyly of Sarcocystis species, although the bootstrap data were inconclusive for some species.

Species-specific identification of Sarcocystis and Toxoplasma by PCR amplification of small subunit ribosomal RNA gene fragments.

The small subunit (SSU) ribosomal RNA (rRNA) genes of four cyst-forming coccidia (Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, Toxoplasma gondii) infecting sheep were analysed for unique target sequences which could be used as priming sites for species-specific polymerase chain reactions (PCR). A total of 11 putatively species-specific oligonucleotides were tested in combination with universal oligonucleotides designed for conserved regions of the SSU rRNA genes of eukaryotes. For each parasite species two of these oligonucleotide pairs were found to be ideal primers for species-specific amplification of SSU rRNA gene fragments. The specificity of the PCR assays was optimised by testing different PCR parameters (annealing temperature, magnesium ion concentration, number of amplification cycles) with genomic DNA preparations derived from S. tenella, S. arieticanis, S. gigantea, T. gondii and sheep. The new PCR assays are a powerful tool for species-specific differentiation of the three ovine Sarcocystis spp. and T. gondii.

The phylogenetic relationships of Kronborgia (Platyhelminthes, Fecampiida) based on comparison of 18S ribosomal DNA sequences.

Approximately 580 bp at the 5' end of the small subunit RNA gene were amplified by PCR for 19 platyhelminth taxa, and Homo and Artemia were used as outgroups. These were analysed to test the hypothesis that fecampiids and Neodermata are sister groups. No evidence was found that the fecampiid Kronborgia isopodicola is closely related to the Neodermata or to the Rhabdocoela (in which the fecampiids are usually included). Morphological, including ultrastructural, characters and DNA data do not support a close relationship of fecampiids with any other platyhelminth taxon, although the DNA sequence analysis provides some evidence that the Acoela and Tricladida are closest. Fecampiids are sufficiently different from any other platyhelminth group to warrant establishment of a class, Fecampiida, for them. A diagnosis of the new class is given.

The phylogeny of Neospora caninum.

Morphological studies by electron microscopy on the protozoan Neospora caninum have shown that this organism possesses a subcellular structure typical of parasites classified in the family Sarcocystidae, subclass Coccidiasina of the phylum Apicomplexa. Using a strategy based on DNA sequence analysis of products derived by asymmetric PCR to determine the nucleotide sequences, we have tested the validity of this classification by comparing the small subunit ribosomal RNA (18S rRNA) gene sequences of N. caninum with those of other parasitic protozoa classified in the phylum Apicomplexa. The results of this analysis confirm the placing of N. caninum in the family Sarcocystidae and place it as a sister group to Toxoplasma gondii in the phylum Apicomplexa.