Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus datasetNucleotide sequence data described in this paper have been deposited in the EMBL database under the Accession number AM177177.. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34–40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.

One of the most fascinating anti-predator responses displayed by parasites is that of hairworms (Nematomorpha). Following the ingestion of the insect host by fish or frogs, the parasitic worm is able to actively exit both its host and the gut of the predator. Using as a model the hairworm, Paragordius tricuspidatus, (parasitizing the cricket Nemobius sylvestris) and the fish predator Micropterus salmoïdes, we explored, with proteomics tools, the physiological basis of this anti-predator response. By examining the proteome of the parasitic worm, we detected a differential expression of 27 protein spots in those worms able to escape the predator. Peptide Mass Fingerprints of candidate protein spots suggest the existence of an intense muscular activity in escaping worms, which functions in parallel with their distinctive biology. In a second step, we attempted to determine whether the energy expended by worms to escape the predator is traded off against its reproductive potential. Remarkably, the number of offspring produced by worms having escaped a predator was not reduced compared with controls.

Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1–4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5–9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.

A molecular approach was used to genetically characterize 5 species (Aoruroides queenslandensis, Blattophila sphaerolaima, Cordonicola gibsoni, Desmicola ornata and Leidynemella fusiformis) belonging to the superfamily Thelastomatoidea (Nematoda: Oxyurida), a group of pinworms that parasitizes terrestrial arthropods. The D3 domain of the large subunit of nuclear ribosomal RNA (LSU) was sequenced for individual specimens, and the analysis of the sequence data allowed the genetic relationships of the 5 species to be studiedNucleotide sequence data reported in this paper are available in the GenBank®, EMBL and DDBJ databases under the Accession numbers: AM232755–AM232763.. The sequence variation in the D3 domain within individual species (0–1·8%) was significantly less than the differences among species (4·3–12·4%). Phylogenetic analyses, using maximum parsimony, maximum likelihood, and neighbour-joining, tree-building methods, established relationships among the 5 species of Thelastomatoidea and Oxyuris equi (a species of the order Oxyurida). The molecular approach employed provides the prospect for developing DNA tools for the specific identification of the Thelastomatoidea, irrespective of developmental stage and sex, as a basis for systematic, ecological and/or population genetic investigations of members within this superfamily.

A cDNA library was constructed from muscle larvae of Trichinella pseudospiralis. A cDNA clone, designated as Tp8 contained a cDNA transcript of 1326 bp length with a single open reading frame, which encoded 303 amino acid residues (34187 Da, estimated molecular mass). The predicted amino acid sequence of the clone had an identity of approximately 60% to the Rcd1 (Required cell differentiation 1) -like proteins among a wide range of organisms. Real-time quantitative polymerase chain reaction results showed that the transcription level of Tp8 gene reached the highest value in adult worms, and that the transcription level in muscle larvae before stichosome formation was higher than in muscle larvae after stichosome formation. The recombinant Tp8 protein migrated at 37 kDa and reacted to antibody against T. pseudospiralis excretory-secretory (E-S) products and sera from mice infected with T. pseudospiralis. An antibody against the Tp8 recombinant protein could stain proteins migrating at approximately 34 kDa (which is the expected size from the sequence) on Western blotting of E-S products from muscle larvae. An immunocytochemical study showed that the Tp8 protein was present within the stichocyte of muscle larvae and adults worms.

cDNAs encoding the immunodominant allergens tropomyosin and paramyosin were amplified from RNA extracted from the sheep scab mite Psoroptes ovis. The tropomyosin cDNA contained an open reading frame (ORF) of 852 bp which encoded a predicted protein with 98% and 97% identity to the house dust mite allergens Der f 10 and Der p 10 respectively. The complete paramyosin ORF generated by RT-PCR was 2625 bp in length and encoded an 875aa predicted protein of 102·6 kDa with 97%, 95% and 89% identity to the paramyosins of Dermatophagoides pteronyssinus (Der p 11), Sarcoptes scabiei and Blomia tropicalis (Blo t 11) respectively. Full length tropomyosin and truncated and full-length paramyosin were expressed as recombinant proteins. IgG and IgE in sera from sheep with a 6-week duration primary infestation of P. ovis did not detect either full-length or truncated recombinant paramyosin. IgG in both infested and naïve sheep sera detected recombinant tropomyosin, suggesting cross-reactivity to tropomyosin and to other invertebrate species to which the sheep may have been exposed. Staining with antibodies directed against tropomyosin and paramyosin was observed throughout sections of P. ovis. Staining was especially prevalent in the anterior sections of the mites, possibly associated with locomotory muscles in this region.

Acetylcholine (ACh) is one of an array of neurotransmitters used by invertebrates and, analogous to vertebrate nervous systems, acetylcholinesterase (AChE) regulates synaptic levels of this transmitter. Similar to other invertebrates, nematodes possess several AChE genes. This is in contrast to vertebrates, which have a single AChE gene, transcripts of which are alternatively spliced to produce different types of the enzyme which vary at their C-termini. Parasitic nematodes have a repertoire of AChE genes which include those encoding neuromuscular AChEs and those genes which code for secreted AChEs. The latter proteins exist as soluble monomers released by the parasite during infection and these AChE are distinct from those enzymes which the nematodes use for synaptic transmission in their neuromuscular system. Thus far, Dictyocaulus viviparus is the only animal-parasitic nematode for which distinct genes that encode both neuromuscular and secreted AChEs have been defined. Here, we describe the isolation and characterization of a cDNA encoding a putative neuromuscular AChE from D. viviparus which contains a tryptophan amphiphilic tetramerization (WAT) domain at its C-terminus analogous to the common ‘tailed’ AChE form found in the neuromuscular systems of vertebrates and in the ACE-1 AChE from Caenorhabditis elegans. This enzyme differs from the previously isolated, D. viviparus neuromuscular AChE (Dv-ACE-2), which is a glycosylphosphatidylinositol-anchored variant analogous to vertebrate ‘hydrophobic’ AChE.

Protozoan cysts and helminth eggs preserved in a coprolite from the Early Cretaceous Bernissart Iguanodon shaft in Belgium demonstrate that representatives of 3 phyla parasitized dinosaurs by that period. These fossil parasite stages are described and their possible effect on dinosaurs discussed. These findings represent the earliest fossil records of protozoan and helminth parasites of terrestrial vertebrates.

The monogenean Gyrodactylus salaris Malmberg, 1957 is an economically important parasite on Atlantic salmon whereas the morphologically very similar G. thymalli Žitňan, 1960 on grayling is considered harmless. Even molecular markers cannot unambiguously discriminate both species. The nuclear internal transcribed spacer (ITS) sequences are identical in both species, and although mitochondrial cytochrome oxidase I (COI) sequences show substantial variation, no support for monophyly of either species is found. Analysis of nucleotide sequences of the intergenic spacer (IGS) have, however, been interpreted as support for 2 species. Here, IGS and COI sequences from 81 G. salaris and G. thymalli specimens of 39 populations across the species' distribution range were determined. Mitochondrial diversity was not reflected in the nuclear marker. Since various 23 bp IGS repeat types usually differ by just one nucleotide and sequences primarily differ in the number and order of repeat types, alignments may be biased and arbitrary, impeding meaningful phylogenetic analyses. The hypothesis that parasites on rainbow trout represent hybrids of both species is rejected. The presence or absence of particular repeat types is not considered informative. We interpret the IGS data as support for G. salaris and G. thymalli being a single species.

The scientific aspect is high calibre and well documented regarding the life-cycle, biology and pathology caused by the eyeworm – Thelazia callipaeda. The studies were well planned and showed that the house fly-Musca domestica was (is) not the intermediate host. On the contrary, it was demonstrated that the first larval stage is capable of developing to the third infective larval stage in the fly genera – Amniota and Phortica. The research is excellent and the narrative on the DVD complements well the science. Commercial aspects of the parasiticides for control of the nematodes are also well presented. The DVD would be of interest to laypersons, in addition to practicing parasitologists, veterinarians and physicians.

The study of host-parasite relationships usually requires reliable estimates of parasite intensity, which is often estimated from parasite propagule concentration in faeces. However, parasite excretion in faeces may be subject to variation due to endogenous or exogenous factors that must be identified to obtain reliable results. We analysed the effect of the hour of sample collection on propagule counts of 2 intestinal parasites infecting the red-legged partridge: the capillarid nematode Aonchoteca caudinflata and coccidia of the genus Eimeria (Protozoa). Also, we test whether there are differences in propagule counts between caecal and intestinal faeces. Individual faecal samples from infected birds were collected daily at 4 different hours during several days. The hour of the day exerted a very strong effect on propagule counts, excretion of both types of parasites showing a clear and constant increase from dawn to dusk. Also, capillarid eggs were more abundant in intestinal than in caecal faeces, whereas the inverse pattern was found for coccidian oocysts. Standardization of the hour of sample collection or statistical control of this variable is recommendable to prevent bias. Similarly, in bird species with long caeca, consistent collection of one type of faeces may avoid significant errors in parasite burden estimates.

Extending a previous investigation of the genotypic variability within Ascaris from humans and pigs, experimental infections of mice and pigs with selected genotypes of Ascaris were performed in the present study to explore possible host affiliation. Initial findings indicate that there is a significant difference in the ability of Ascaris eggs of genotype G1 (derived from human) and G3 (derived from pig) to infect and establish as larvae in mice and as adults in pigs, supporting the difference in the frequencies of the genotypes reported previously in natural Ascaris populations in pigs and humans in areas in China endemic for ascariasis.