Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70·8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4·8. The enzyme was both heat (>37 °C) and cold (<0 °C) labile with an optimal activity at pH 7·2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

Clinical data on the use of artesunate combined with mefloquine in a variety of treatment regimens and parasite loads in Thailand were modelled on the basis of experimentally determined pharmacokinetic data. The model assumed no pharmacodynamic interaction between artesunate and mefloquine, but that the parasites were already resistant to mefloquine. Predictions of the model accorded well with the data. In particular, in accordance with clinical observations, the model showed that monotherapy with either drug failed to cure at moderate parasitaemia, yet such patients could be treated effectively with the combination of 3 days of artesunate + mefloquine. For high levels of parasitaemia, 5 days of artesunate+mefloquine were needed. Simulations were also performed for situations of lower resistance to mefloquine and for the immune human populations found in Africa. The importance of mathematical modelling of combination therapy is borne out by this study and suggests its wider application for other drug combinations.

Two cases of skin lesions similar to those caused by Leishmania parasites have been reported from Martinique. Parasites isolated from these lesions were unlike Leishmania reference strains by isoenzyme analysis and electron microscopy and were assumed to be monoxenous trypanosomatids which normally only infect invertebrates. Both strains have now been retyped by isoenzyme analysis and found to be identical to each other and distantly related to all other Leishmania species. The sequence of the 18S ribosomal RNA gene and partial sequences of the DNA polymerase alpha and RNA polymerase II largest subunit genes were obtained. These sequences indicated that the Martinique parasites clustered with L. enriettii and were basal to all other euleishmania. However, support for both the position basal to all euleishmania and the clustering with L. enriettii was low. The Martinique parasites may cluster with L.(Leishmania) or L.(Viannia) or form a novel clade within the euleishmania either with or without L. enriettii.

Most of our knowledge about the process of penetration of skin, by cercariae of Schistosoma mansoni, has been gained from studies carried out in vivo with laboratory animals. Human skin is significantly different from that of other animals but there are obvious practical difficulties in directly studying attachment and penetration with human skin. Techniques have been developed which enable a 3-dimensional ‘skin equivalent’ to be grown in tissue culture, made from different types of human skin cells. The aim of the present study was to investigate cercarial interactions with confluent cultures of the individual skin cell types that make up normal human skin and which will be used to construct a multi-component model. Cercariae behaved differently towards the various cell types tested. They responded least to monolayers of endothelial cells and most to primary keratinocytes, derived from human foreskin and differentiated at an air/liquid interface. This study demonstrates, therefore, that cercariae are capable of distinguishing between different types of skin cells and they preferentially attach to differentiated cells which form the epidermis.

Mathematical models often propose that within-host competition between parasites can be a major factor in the evolution of increased parasite virulence. Kin selection predicts that as the coefficient of relatedness between infecting parasites decreases, the benefits of competition to individual genotypes increases. Thus where parasites can adjust their behaviour in response to current conditions, higher virulence is predicted in multiple genotype infections. There is limited experimental data, however, regarding the effects of mixed strain infections on host and parasite fitness. We investigated, for a snail–schistosome system, whether a conditional increase in replication rates occurred in mixed genotype infections and resulted in increased virulence. Four groups of Biomphalaria glabrata snails were exposed to 1 or 2 laboratory strains of Schistosoma mansoni. Mixed genotype infections were observed to be more virulent than single genotype infections, in terms of reductions in host reproductive success and survival. Parasite reproductive rate was also increased in mixed strain groups. Reduced host reproductive success was suggested to be directly due to the genetic heterogeneity of the parasitic infections resulting in increased host defence costs. Reduced host survival was consistent with an adaptive conditional parasite response.

This paper adds new insight to a molecular phylogeny of Gyrodactylus, based on a complete sequence of the ITS rDNA region of 4 subgenera and a more detailed molecular analysis. We propose a hierarchical approach in elucidating the phylogeny of this species-rich genus. A total of 37 sequences (915–1239 bp) from 10 representative species from 4 out of 6 subgenera, as defined by Malmberg (1970), are included in the analysis. Genetic differences observed at the 5.8S locus provide objective criteria to separate (sub)genera, while deep genetic differences of the spacers form a sound basis for species-specific identification. We demonstrate that each Gyrodactylus subgenus possesses a unique sequence of the 5.8S gene. Thus, there is concordance between the 5.8S gene and the excretory system used by Malmberg (1970) as a diagnostic character of subgenus status. At the species level, there is a discrepancy between morphological and molecular variation. Whereas the morphological variation, expressed in the shape and size of the attachment apparatus, is very low, the molecular variation, expressed at the ITS1 and ITS2 regions, is very high. This can be attributed either to the fast evolving ITS region or to the fact that the genus consists of groups of a higher taxonomic level than previously recognized. Perspectives and limitations of using the ITS rDNA region for a molecular phylogeny of this genus are discussed.

Viviparous gyrodactylid monogeneans which are detached from their host may form an important source of infection in fish communities. This is particularly significant for these ectoparasites which have no specific free-living stage and where transmission usually occurs through transfer of adult parasites when fish come into close contact. In this study, in vitro survival of Gyrodactylus gasterostei was correlated with changes in tissue ultrastructure of parents and their embryos during detachment and following return to a host. At 10 °C, detached worms survived for up to 89 h (50% survival = 20 h) with mortality influenced by both age-independent and age-dependent factors. The gastrodermis of attached, fed parasites is syncytial and contains a variety of vesicles and feeding vacuoles. However, following detachment and starvation, degenerative changes occur within the gastrodermis, eventually leading to marked damage to the embryo/s. When starved worms reattached to a fish, they began browsing on host epithelial cells within 1 min and ultrastructural evidence for phagocytic activity in the gastrodermis was detected after 5 min. Putative waste vacuoles increased in abundance after 5–30 min on the new host, coinciding with the reappearance of electron-dense vesicles and possibly indicating completion of the first intracellular cycle of digestion. Parental feeding directly influenced the normal cyclical maturation and regression of the uterine lining between births. In extreme cases, starvation led to detached parasites (6·1%) aborting their embryos. However, even short periods off the host influenced development and survival of embryos, suggesting that temporary interruption of nutrient flow to the embryo can significantly affect gyrodactylid reproductive rates.

Variation in host susceptibility causes significant differences in infection rates between hosts living in a semi-natural situation. Such knowledge has implications for population dynamics and evolutionary models of host–parasite interactions as well as for estimations of parasite abundance. Infection rates by Lernaeocera branchialis (L.) were measured through time and space on caged Atlantic cod (Gadus morhua L.). One group of hosts, identified by their infection history, developed significantly higher infection rates than the others. These were fish which had been infected previously, but had lost their infection. Differences between groups were consistent through both time and space. Two types of cod seem to have been present in the caged population; a small group of inherently susceptible fish, which were infected, and reinfected if the parasite was lost, and another group of resistant hosts with a small chance of becoming infected.

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

Information on the ultrastructural details of fertilization in filarial nematodes are still unavailable. Here we report new data on this process in Dirofilaria immits, the heartworm of dogs and cats. Electron microscopy allowed us to observe oocytes engulfing spermatozoa through an endocytosis-like process. We also observed spermatozoa inside the oocytes which still possessed their plasma membrane and which were clearly enveloped by a further membrane, likely derived from the endocytosis process. At this stage, at the interface between the sperm membrane and the endocytotic membrane (vacuolar space), we observed flocculent material in the proximity of the membranous organelles (MOs) of the sperm. In the proximity of the MOs, we also observed the enlargement of the vacuolar space. Other images showed the dissolution of the sperm membrane, and the release of nuclear masses and organelles in the egg cytoplasm. We did not observe the fusion of lysosomes to the endocytotic vacuoles. In addition, the lysis of the sperm organelles has never been observed inside the vacuoles containing the whole sperm. Thus we suggest that the degradation of the endocytotic and sperm plasma membranes is determined by material released by the MOs. Since we did not observe the entry of sperm into the oocytes by other mechanisms, we also suggest that endocytosis is the normal process used by the spermatozoon to get into the egg cytoplasm in D. immitis. Finally, during our observations of the seminal receptacle we did not observe any structure in the spermatozoa which could be interpreted as an intracellular bacterium. This is consistent with previous results indicating that the bacterium Wolbachia in filarial nematodes is not transmitted through the sperm.

Unlike other members of the genus, Echinococcus granulosus is known to exhibit considerable levels of variation in biology, physiology and molecular genetics. Indeed, some of the taxa regarded as ‘genotypes’ within E. granulosus might be sufficiently distinct as to merit specific status. Here, complete mitochondrial genomes are presented of 2 genotypes of E. granulosus (G1–sheep-dog strain: G4–horse-dog strain) and of another taeniid cestode, Taenia crassiceps. These genomes are characterized and compared with those of Echinococcus multilocularis and Hymenolepis diminuta. Genomes of all the species are very similar in structure, length and base-composition. Pairwise comparisons of concatenated protein-coding genes indicate that the G1 and G4 genotypes of E. granulosus are almost as distant from each other as each is from a distinct species, E. multilocularis. Sequences for the variable genes atp6 and nad3 were obtained from additional genotypes of E. granulosus, from E. vogeli and E. oligarthrus. Again, pairwise comparisons showed the distinctiveness of the G1 and G4 genotypes. Phylogenetic analyses of concatenated atp6, nad1 (partial) and cox1 (partial) genes from E. multilocularis, E. vogeli, E. oligarthrus, 5 genotypes of E. granulosus, and using T. crassiceps as an outgroup, yielded the same results. We conclude that the sheep-dog and horse-dog strains of E. granulosus should be regarded as distinct at the specific level.