The microsporidian Encephalitozoon cuniculi can infect numerous mammals, including man. Three strains of E. cuniculi have been identified so far, the major marker being the number of a tetranucleotide repeats in the rDNA internal transcribed spacer. We investigated diversity at the chromosomal level through the electrophoretic karyotypes obtained from 15 E. cuniculi isolates from 5 different host species. All preparations provided patterns with 9–12 bands within a narrow molecular size range. Six karyotype forms were distinguished, involving subdivision of strain I into 3 types (A, B, C) and strain II into 2 types (D, E). The types A, B and C were mainly associated with isolates from rabbits of different geographical origins. The types D, E and F were characterized by a reduced chromosome size range, 2 of these appearing specific to a carnivorous host species (D in dog and F in blue fox). Hybridization experiments showed that all E. cuniculi isolates possess 11 chromosomes, with a size polymorphism entailing occasional electrophoretic comigration of heterologous chromosomes and differential migration of homologous ones. DNA rearrangements should occur during mitosis and the hypothesis of diploidy for the basic state of E. cuniculi seems likely.

The microsporidial species Enterocytozoon bieneusi is found among immunocompromised, particularly HIV-infected, patients with chronic diarrhoea, and rarely also among immunocompetent persons with self-limited diarrhoea. Only recently, E. bieneusi was detected in 4 pigs in Switzerland raising the question of a potential zoonotic nature of this parasite. We examined faecal samples of 109 pigs, 24 cows, horses and red foxes each for the presence of E. bieneusi by PCR and compared these isolates with isolates obtained from stool samples of 13 HIV-infected patients living in Switzerland. In animals, E. bieneusi was only identified in pigs with a prevalence of 35%. Analysis of the rDNA internal transcribed spacer (ITS) sequence allowed the classification of E. bieneusi from 28 pigs into 4 distinct genotypes which grouped very closely (identity 96·3–98·8%) together with 2 of the 3 human-derived E. bieneusi genotypes. Hence, E. bieneusi seems to be a common parasite in swine, but no genotypes were identified that were found in humans. Nevertheless, swine might serve as a new animal model for enterocytozoonosis.

Promastigotes of Leishmania major were frequently detected in the urine droplets discharged by infected Phlebotomus papatasi and P. duboscqi females during feeding. Parasites were present in the urine of 37·5% P. papatasi and 16·1% P. duboscqi females, even in those with low intensity gut infections. Free-swimming forms (elongated nectomonads, short slender promastigotes and metacyclic forms) predominated in excreted droplets. Viability of excreted parasites was proved by cultivation on blood agar, and the presence of metacyclic forms in urine droplets was confirmed by specific fluorescence assay with 3F12 antibodies. While the release of promatigotes from the anus of the sandfly was frequent, these were rarely egested from the mouth-parts of sandfly females (1·3% for P. duboscqi and 0% for P. papatasi) fed on microcapillaries, even if the females were heavily infected. The possible role and significance of the discharge of parasites in sandfly urine are discussed.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.

Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.

The formation and development of monogenean communities on the gills of roach fry was followed in 1992 from early June to October (size range 9 to 47 mm). Roach fry (n=291) were sampled weekly from the small, humic River Rutajoki in central Finland. A further 209 roach fry were reared in a fish farm supplied by water from the river. Four Dactylogyrus species were found: D. nanus, D. crucifer, D. micracanthus and D. suecicus. Other species found on the gills were Gyrodactylus sp. and Paradiplozoon homoion. The first Dactylogyrus juvenile occurred on a 12 mm long fish fry in late June and the first adult (D. nanus) 1 week later in Tank 1. D. nanus was also the most common parasite in the river. Young fry had high numbers of Gyrodactylus sp. on the gills compared with adult roach in previous studies. Signs of site preference were found; D. nanus and D. micracanthus preferred the 2nd gill-arch and Dactylogyrus juveniles the outmost one. Abundances of monogenean infracommunities increased until the middle of August when, on average, 1·9 species and 3·7 worms per fry were noted and decreased then as the water temperatures fell. A lack of competition between species is suggested; for example, D. crucifer appeared on the gills at that time when abundance of D. nanus was at its highest. The roach fry hatched and reared in the fish farm revealed that larvae of the most common dactylogyrid species were able to disseminate and colonize over 120 m from the river to the farm. Influence of change colonization events was shown by the appearance and development of Gyrodactylus sp. population on roach fry in only 1 of the 2 tanks at the farm.

The excretory–secretory (E–S) products of the parasitic nematodes Trichostrongylus colubriformis and Nematodirus battus were found to modify the in vitro proliferation of the tumorous colic HT29-D4 cell line of epithelial origin. A characteristic feature of these E–S products is the presence of a high level of acetylcholinesterase (AChE) activity, the biological significance of which remains unclear. To determine a possible role of AChE on cell growth, the enzyme was purified from E–S products using edrophonium chloride. Purity was confirmed by polyacrylamide gel electrophoresis, using silver and Karnovsky stains, before assessing its effects on cell proliferation. The purified AChE was incorporated at different concentrations in a culture medium of HT29-D4 cells. A mitogenic effect was shown for low concentrations (0·1–14 units). By contrast, an inhibitory effect was noted at high concentrations (35–1400 units). Furthermore, polyclonal antibodies were prepared and depletion of AChE in E–S products by immunoprecipitation or affinity chromatography resulted in a partial or total disappearance of the stimulatory effect of cell growth. Thus, the results from this in vitro study suggest a modulatory role for AChE secreted by nematode parasites on the proliferation of epithelial cells of the host.

Many studies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) have reported that only a small proportion (typically <40%) of infective stages (dauers), even under apparently ideal conditions, actually infect a host. The ‘phased infectivity hypothesis’ is most frequently invoked to explain this pattern of low infection with entomopathogenic nematodes. It proposes that at a given point in time not all individuals are infectious i.e. infectiousness is delayed in some individuals. We tested experimentally several predictions based on this hypothesis. Specifically, if phased infectivity occurs, we should be able to expose dauers to increasing numbers of potential hosts until dauers no longer infect and still be able to recover viable dauers. These recovered dauers which did not infect should be infectious at some later point in time. However, our results do not support the phased infectivity hypothesis for 3 species of Steinernema: most dauers could be recovered in one sampling round when provided with sufficient suitable hosts. In contrast, Heterorhabditis bacteriophora frequently did not infect all available hosts, and infectious dauers were recovered in subsequent sampling rounds. This result is more consistent with the phased infectivity hypothesis, but further research is needed before we can be more confident in the hypothesis. For all species tested, the number of available hosts influenced population levels of nematode infectivity. This suggests that the infection status of hosts can influence whether a dauer infects. Our results indicate that phased infectivity is not a common phenomenon in entomopathogenic nematode dauers, despite the widespread acceptance of this hypothesis.

The surface coat of the infective larvae of the parasitic nematode Trichinella spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surface probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron microscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity of PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4°C, suggesting the shedding process required metabolic energy. Pre-labelled, unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated failed to re-label with the probe, suggesting that activation provides an irreversible trigger for surface changes. PKH26, therefore, is a useful marker for larval activation. Examination of the shed coat material by scanning electron microscopy revealed 2 types of morphologies; one comprising thin multilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat material demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treatment and a 47–60 kDa set of protein(s). Analysis of the surface lipids by electrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14[ratio ]2) and an unidentifiable component of 339·4 Da. These two lipids constituted 36·9% and 36% by mass of surface coat lipids respectively. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48[ratio ]4), PE (C40[ratio ]4 and C38[ratio ]4), PS (C40[ratio ]4), lysoPC (C20[ratio ]2 and C18[ratio ]2) and lysoPA (C14[ratio ]2). These observations are discussed with respect to the role of the surface coat and its shedding in the T. spiralis host–parasite relationship.

Polymerase chain reaction–ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR–RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the camel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.