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Mitochondrial genome sequences provide useful markers for investigating population genetic structures because of their maternal inheritance and high evolutionary rates. There is, however, a paucity of information on mitochondrial genomes for many parasitic organisms, including nematodes, which appears to relate mainly to technical limitations and (for modestly funded laboratories) the cost associated with full mitochondrial genome sequencing. In this article, we describe a simple, relatively inexpensive long-PCR approach for the amplification (using two sets of primers) of the entire mitochondrial genome from individual parasitic nematodes for subsequent sequencing, which overcomes these limitations. We employed two species of human hookworm (Ancylostoma duodenale and Necator americanus; order Strongylida) to establish the long-PCR conditions, and then extended its use to a number of other species of parasitic nematode of the class Secernentea (orders Strongylida, Ascaridida and Rhabditida). The long-PCR method for the amplification of the entire mitochondrial genome from single nematodes, coupled with direct sequencing of amplicons, provides a useful tool for the comparative analysis of genome organisation and evolution of a range of nematode groups. It also creates a platform for molecular ecological and population genetic studies., (Copyright 2002 Elsevier Science Ltd.)

The evolutionary relationships of 21 species of trichostrongyloid nematodes were determined by use of sequence data of the second internal transcribed spacer of the ribosomal DNA aligned according to secondary structure information. Irrespective of the method of analysis used, the topologies of the phylogenetic trees derived from the molecular data differed with respect to all four hypotheses proposed previously for the evolutionary relationships of the different subfamilies within the Trichostrongylidae based on morphological data. Thus, the molecular data set did not resolve the conflict between the four previous proposals for the subfamilial relationships. Nonetheless, all trees derived from the molecular data showed strong support for the exclusion of the genera Filarinema and Amidostomum from the clade containing the species within the family Trichostrongylidae. This represents a major difference from the most recent proposal of the systematics of the Trichostrongyloidea in which these two genera were included within the Trichostrongylidae. Therefore, the molecular data support an earlier systematic framework in which Filarinema and Amidostomum were considered to be sister groups of the Trichostrongyloidea., (Copyright 2001 Academic Press.)

Necator americanus and Ancylostoma duodenale are the two most important species of human hookworm, and occur in sympatry over much of their distribution. The specific diagnosis of hookworm infections is central to control. Diagnosis currently relies on the detection of hookworm eggs in human faeces and/or the specific identification of larvae by 'copro-culture' combined with microscopic examination. However, the eggs of the two species are morphologically indistinguishable, and the procedure of copro-culture is tedious and time-consuming to carry out. To work toward overcoming these limitations, a molecular approach utilizing genetic markers in the first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was established. The ITS-1 sequences of both hookworm species were determined, and specific oligonucleotide primers designed to regions of major sequence difference between the species were evaluated in polymerase chain reaction (PCR). Using a range of control samples, the primers allowed the specific identification of as little as 10 pg DNA of A. duodenale or N. americanus. The findings indicate clearly the potential for specific PCR to confirm the identity of eggs from faeces and larvae from the environment or host tissues. This should have important implications for studying fundamental aspects relating to anthelmintic efficacy and the epidemiology of hookworms.

Oesophagostomiasis in humans due to infection with Oesophagostomum bifurcum (nodular worm) is of major human health significance in northern Togo and Ghana where Necator americanus (human hookworm) also exists at high prevalence. However, very little is known about the transmission patterns of O. bifurcum, partly due to the difficulty in differentiating O. bifurcum from N. americanus at some life-cycle stages using morphological features. To overcome this limitation, a molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to the regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR assays were developed for the specific amplification of DNA of O. bifurcum or N. americanus, which have the potential to confirm the identity of eggs from faeces and larvae from the intestine or environment. The application of species-specific PCR has important implications for studying the epidemiology and population biology of O. bifurcum.

At some stages of development, it is impossible to identify the porcine nodular worms Oesophagostomum dentatum and O. quadrispinulatum to the species level using morphological parameters. A molecular approach utilizing genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA was developed to overcome this limitation. The ITS-2 sequence of each species was determined, and specific oligonucleotide primers were designed to regions of greatest sequence difference between the species. Utilizing these primers, rapid PCR procedures were developed for the specific amplification of DNA of O. dentatum or O. quadrispinulatum, which are now used routinely to monitor the purity of larval cultures and to confirm the identity of larvae derived from the intestine or faeces. The application of specific PCR has major implications for studying the population biology of nodular worms in the pig model.

Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosomal DNA was amplified from genomic DNA by polymerase chain reaction (PCR) using conserved primers, digested separately with six restriction endonucleases (AluI, BfaI, CfoI, Hae III, VSpI and XbaI) and the fragments separated by agarose gel electrophoresis. The PCR products of the three Strongylus species were approx. 90-100 bp smaller in size compared with those of the other 13 species. The PCR-RFLP analysis of the rDNA region spanning the first and second internal transcribed spacers plus the 5.85 rDNA gene (ITS+) produced characteristic patterns for each of the 16 species examined, and no variation in RFLP patterns was detected within the species Cy. catinatum, where multiple isolates were analysed. The study demonstrates that the internal transcribed spacer sequences provide genetic markers for the species identification of a range of equine strongyles. These markers will be of use for the identification of egg and larval stages, where morphological characters alone are unreliable. The results also indicate that the spacer sequences will be of use to study the systematics of equine strongyles.