The mouse major histocompatibility complex (MHC) class I sequence was detected in 8-week-old Schistosoma mansoni by in situ polymerase chain reaction (in situ PCR). The signals to the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of S. mansoni. Signals were also observed in the cytoplasm of the tegumental tubercles. This finding suggested the possibility of MHC gene transfer from the host to schistosomes. Furthermore, the class I MHC sequence was detected in the DNA extracted from the cercariae of S. mansoni by nested PCR. Neither the nucleotide sequence of class I MHC detected in adult worm DNA nor that of class I MHC detected in the host (mouse) DNA was identical with that of class I MHC detected in the cercarial DNA. From the data we assumed that S. mansoni may have retained their own mouse class I MHC sequence in their genome throughout their life-cycle.

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.

We test the hypothesis that living on larger fish may impose constraints, i.e. the need to develop large attachment organs, related to the necessity to remain attached on large gills. For this, we compiled data on body size and morphometric measurements of attachment organs of 44 Dactylogyrus species (ectoparasites with direct life-cycle) from 19 cyprinid species. Nineteen dactylogyrid species were considered as specialists (infecting only 1 host species) and 25 as generalists (infecting more than 1 species). The lack of phylogenetic information lead us to perform comparative analyses using raw values and independent contrasts obtained by random phylogenies. Our results show that rich parasite communities are formed by specialists and generalists whereas poor communities are composed mainly of generalist parasites. Moreover, specialists are found on larger hosts, which may reflect a specialization on a predictable resource, as larger fish live longer and offer large gills for parasite colonization. Parasite specialization is shown to be linked with adaptation of attachment organs to their fish hosts. Two morphometric variables of the attachment organ, the total length of anchor and length of base of anchor, were positively correlated with host length for specialists.

An epidemiological study on dicrocoeliosis caused by Dicrocoelium dendriticum was carried out on sheep, molluscs and ants in the mountains of León province (NW Spain) between 1987–1991. The results concerning the intermediate hosts and a review of some aspects of dicrocoeliosis are summarized. Mollusc collection for the helminthological study was random throughout the study area at fortnightly intervals. Twenty-nine Gastropoda species were identified. D. dendriticum infection was only detected in 2·98% of the 2084 Helicella itala examined and in 1·06% of 852 H. corderoi. The highest infection prevalence was detected in H. itala in September and in H. corderoi in February. Daughter sporocysts with well-developed cercariae predominated in spring and autumn. Infection prevalence increased with mollusc age and size. Ants were collected from anthills or plants to which they were attached. The behaviour of ants in tetania was followed. Twenty-one Formicidae species were identified, but only the following harboured D. dendriticum: Formica cunicularia (1158 examined specimens, 0·69% infection prevalence, 2–56 metacercariae per ant); F. sanguinea (234, 1·28%, 2–63); F. nigricans (1770, 4·97%, 1–186); F. rufibarbis (288, 6·59%, 2–107). In a flat area close to León town, 95·39% of the 2085 F. rufibarbis specimens collected in tetania contained metacercariae (1–240) in the abdomen. These were used for parasite characterization by isoelectric focusing and to infect lambs and hamsters. Only one brainworm per ant was found.

A 186 bp Echinococcus granulosus-specific repetitive element, TREg, was used to assess genetic variation between strains. In G7 genotype (pig strain) it has the characteristics of a satellite DNA element with a copy number of 23000 per haploid genome. Analysis, by sequencing of TREg monomers, showed a great degree of identity within them. In the G1 genotype (common sheep strain) TREg-like repetitive elements were found in an interspersed distribution throughout the genome and in only 120 copies. The sequences of these monomers showed a great degree of variation between them and with TREg of G7 origin. The G6 genotype (camel strain) showed a pattern of distribution and copy number similar to the G7 genotype, and the G2 genotype (Tasmanian sheep strain) similar to the G1 genotype. Isolates from the G5 (cattle strain) and G4 (horse strain) genotypes also showed unique hybridization patterns in Southern blot experiments. The genomic plasticity of E. granulosus, which may have important consequences in the epidemiology and control of cystic hydatid disease is reflected in the results of this work.

Analysis of natural populations of Trypanosoma brucei has shown that there is linkage disequilibrium between alleles at pairs of loci in isolates taken from the field. This disequilibrium can occur as a result of a low frequency of genetic exchange, the masking of frequent genetic exchange by the rapid expansion of a few genotypes or by the treatment of 2 (or more) genetically isolated populations as a single population. We have analysed stocks from 2 geographically separate locations using 3 minisatellite markers to determine the frequencies of the alleles in each area and the frequency and nature of the multilocus genotypes. The results show that many alleles and multilocus genotypes are unique to each geographical location, supporting the conclusion that these populations are genetically isolated with limited or no gene flow between them. This geographical substructuring needs to be taken into account in considering the origins of the linkage disequilibrium in a number of populations.

The red poultry mite, Dermanyssus gallinae, is one of the most economically deleterious ecto-parasites of layer hens worldwide. D. gallinae is difficult to eliminate from infested poultry farms, and even to study, because it resides on the host only during the bloodmeal at night, and hides in the crevices of poultry houses during the day. Here, the life-cycle of D. gallinae was reproduced entirely in vitro. Mites were incubated in a glass pipette at 30 °C, 60–95% relative humidity and total darkness. A feeding apparatus, composed of a membrane, reservoir and blood was fitted on the pipette during bloodmeals. We tested feeding rates on blood mixed with 1 of 3 anti-coagulants (EDTA, heparin and trisodium citrate) at different concentrations, and biological and artificial membranes. The best engorgement and survival rates for all 3 haematophagous life-stages of the parasite were observed in 1-day-old chick membranes and heparinized (0.02 mmol/ml) blood. We then describe the steps in developing a complete self-sustaining in vitro life-cycle. A colony of mites was maintained in vitro for 7 generations. Losses in the first generation were heavy, but survival had multiplied 5-fold by the fifth generation. We hypothesize that heavy mortality rates during the first life-cycle correspond to selective pressure: only the mites which fed and survived in vitro were able to reproduce.

The avian schistosomatid Trichobilharzia ocellata plays an important role as causative agent of cercarial dermatitis of humans in Europe. In order to improve the taxonomic knowledge on this parasite, studies of miracidial chemo-orientation as well as experimental infections of different snail species were conducted using strains of T. ocellata and T. franki. Both schistosomes exhibited a high intermediate host specificity. The miracidia clearly preferred the SCW (snail-conditioned water) of the respective natural intermediate host to SCW of other sympatric snail species. T. ocellata proved to be capable of infecting Lymnaea stagnalis and Stagnicola palustris, but could not develop in Radix ovata or R. auricularia. T. franki established an infection in specimens of R. auricularia and R. ovata, but not in L. stagnalis or S. palustris. The results imply that the intermediate host spectrum of T. ocellata is limited to L. stagnalis and S. palustris. Findings of T. ocellata (or Cercaria ocellata) that originated from snails of the genus Radix are likely to have actually belonged to species such as T. franki or T. regenti. The assumption that T. szidati is synonymous to T. ocellata is also discussed.

The purpose of this study was to determine under laboratory conditions over a period of 311 days if infection with the nematode Anguillicola crassus induces stress in European eels (Anguilla anguilla), and stimulates the endocrine stress axis as measured by serum cortisol levels. Eels were experimentally infected with 3rd-stage larvae (L3) in different doses to simulate natural conditions with varying infection pressures. Blood samples were drawn from the caudal vein every 2 weeks and serum cortisol concentrations were determined by radioimmunoassay (RIA). The results showed that the application of L3 resulted in a significant increase in the cortisol levels. The period of time at which elevated cortisol values were observed was consistent with the time of larval development and the appearance of adult A. crassus. Thus, there is a stress response to the larval and young adult stages, but no chronic response to older adults. Therefore, it is likely that infection of eels under natural conditions especially with a high number of larvae may be a considerable stressor, in combination with different environmental factors like water temperature, pH, oxygen concentration, pollution and inter-individual relationships.

Pasteuria penetrans is a Gram-positive endospore-producing bacterium that is a parasite of root-knot nematodes. Attachment of endospores to the cuticle of the nematode is the first stage in the infection process. Western blot analysis with monoclonal and polyclonal antibodies that recognize the 30 kDa heparin-binding domain (HBD) and the 45 kDa gelatin-binding domain (GBD) fragments of human fibronectin (Fn) revealed a series of polypeptides of approximately 40, 45 and 55 kDa present in crude cuticle extracts of Meloidogyne javanica 2nd-stage juveniles. The results suggest that the structure of the nematode fibronectin is different to the fibronectins so far characterized. Pre-treatment of endospores of Pasteuria with either the HBD or the GBD was found to inhibit binding to the nematode cuticle. The larger GBD fragment was the most effective at blocking adhesion. Pre-treatment of the GBD fragment with gelatin prevented the GBD fragment from inhibiting endospore attachment to the nematode cuticle.

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

Fascioliasis due to the digenean species Fasciola hepatica has recently proved to be an important public health problem, with human cases reported in countries of the five continents, including severe symptoms and pathology, with singular epidemiological characteristics, and presenting human endemic areas ranging from hypo- to hyperendemic. One of the singular epidemiological characteristics of human fascioliasis is the link of the hyperendemic areas to very high altitude regions, at least in South America. The Northern Bolivian Altiplano, located at very high altitude (3800–4100 m), presents the highest prevalences and intensities of human fascioliasis known. Sequences of the internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA of Altiplanic Fasciola hepatica and the intermediate snail host Lymnaea truncatula suggest that both were recently introduced from Europe. Studies were undertaken to understand how the liver fluke and its lymnaeid snail host adapted to the extreme environmental conditions of the high altitude and succeeded in giving rise to high infection rates. In experimental infections of Altiplanic lymnaeids carried out with liver fluke isolates from Altiplanic sheep and cattle, the following aspects were studied: miracidium development inside the egg, infectivity of miracidia, prepatent period, shedding period, chronobiology of cercarial emergence, number of cercariae shed by individual snails, survival of molluscs at the beginning of the shedding process, survival of infected snails after the end of the shedding period and longevity of shedding and non-shedding snails. When comparing the development characteristics of European F. hepatica and L. truncatula, a longer cercarial shedding period and a higher cercarial production were observed, both aspects related to a greater survival capacity of the infected lymnaeid snails from the Altiplano. These differences would appear to favour transmission and may be interpreted as strategies associated with adaptation to high altitude conditions.

Thirty Holstein Friesian heifer calves, 3 months of age, were used to evaluate whether a low primary infection with Dictyocaulus viviparus would result in immunity against challenge infection 5 or 12 weeks later. Fifteen calves were experimentally infected with 30 larvae at day 0, while the other calves remained as uninfected controls. On day thirty-five 6 primary infected (G1) and 6 non-infected calves (G2) were challenged with 2000 larvae. These groups were necropsied on day 70. On day 84 the remaining 9 primary infected (G3) and 9 non-infected (G4) calves were similarly challenged and necropsied on day 119. Respiratory signs developed in most calves from approximately 2 weeks after challenge, but signs were more severe in G2 and G4 than in G1 and G3. The low primary infection resulted in significantly reduced faecal larval excretion (>80%) and worm burdens (>70%). In the primary infected groups the proportion of female worms in the worm burden was increased, suggesting that part of the developed immunity affected survival of the worms after their establishment in the host. Worm lengths and worm fecundity were also significantly reduced. All observed effects were stronger following challenge on day 84 than on day 35, but the effect of the duration of a primary infection (contrast between G1 and G3) was only statistically significant in the case of mean faecal larval excretion on the day of necropsy (P<0.05) and fecundity (P<0.05).

In order to identify antigens associated with protection and those associated with active infection, the humoral immune response of 6 Mandrillus sphinx immunized with 150 irradiated L3 and challenged with 100 normal L3 of Loa loa or 6 animals infected with 100 L3 were compared. The plasma of these animals was analysed by Western blot using adult, Mf and L3 antigens. Several antigens with molecular weights varying from 120 kDa to 13 kDa were recognized by the plasma of all animals. It was shown that early recognition of microfilarial antigens with molecular weights of 97, 68, 45 and 33 kDa correlated with the amicrofilaraemic state. A total of 83% of animals with circulating microfilariae had antibodies against the microfilariae 21 kDa antigen. Furthermore, the antibodies against the 21 kDa appeared 1 month before detection of microfilariae in the peripheral blood of 80% of these animals, and declined when animals became amicrofilaraemic. In contrast, when L3 antigen was used, a molecule with a relative molecular weight of 20 kDa was recognized by antibodies of the only animal which remained amicrofilaraemic for 1 year after immunization with irradiated L3. These results suggest that the microfilarial molecule of 21 kDa may be useful as a marker of Loa loa patent infection, whereas the 97, 68, 45 and 33 kDa molecules of microfilariae and the L3 molecule of 20 kDa may be associated with resistance against Loa loa.

We studied variation in the structure of component communities of helminths in sand lizards, Lacerta agilis, from 30 localities in the Ukraine and Bulgaria. Thirty-five separate samples of lizards, with a total of 661 completely censused infracommunities, yielded 30 helminth species (4 cestodes, 10 trematodes, 3 acanthocephalans and 13 nematodes). In its range within the Ukraine, L. agilis serves as the final host for 13 species of which only 3 (S. lacertae, S. hoffmanni and P. molini) can be considered as lizard specialists. A characteristic feature of these helminth component communities was the large proportion of heteroxeneous helminth species for which L. agilis serves as paratenic host. Sand lizards in the meadow steppeland zone were primarily parasitized by larval helminths that represented a major proportion of the total number of all worms recovered while those sampled in the grassland/forest transition zone were characterized by substantially higher proportions of adult helminths using lizards as final hosts. However, L. agilis was parasitized by a much higher proportion of lizard specialists in the ‘typical' habitats of the meadow steppeland zone as opposed to those located in ‘marginal' habitats in the grassland/forest transition zone, where helminths were shared to a greater extent with amphibian hosts.

Parasite aggregation within the gill arches of 1 host species (Rutilus rutilus L.) was studied in the case of 9 congeneric monogeneans belonging to the genus Dactylogyrus. Both intra- and interspecific aggregation were followed by considering gill arches as independent patches. Parasite species were found to be aggregated both intra- and interspecifically. We showed that the intensity of parasite infection was positively related to intraspecific aggregation only for the more abundant species. No relationship was found between intensity of parasite infection and niche size when correcting for total parasite intensity. This may suggest that when parasite intensities are low, intraspecific competition may not have a strong effect on parasites. Conversely, when looking at the evolution of niche size and intraspecific aggregation for each species separately, intraspecific aggregation decreased for the most abundant species (D. crucifer) suggesting that this species is more competitive than others when total parasite intensity increases. When considering interspecific aggregation, Dactylogyrus species were found to be positively and negatively aggregated. Following the prediction of morphological and ecological similarity for congeneric species, Dactylogyrus species similar in morphometry of attachment parts tend to be more positively aggregated among their gill microhabitats than Dactylogyrus species with morphometric differences in attachment parts.

We tested the null hypothesis that populations of hosts trapped in isolated neighbouring locations showing comparable habitat quality, should support similar helminth parasite communities. The study was undertaken in a 2-week period in late summer in NE Poland in a single year, thereby eliminating seasonal and between-year variation in parasite burdens. A total of 139 Clethrionomys glareolus (bank vole) were sampled from 3 forest sites of similar habitat quality. Total species richness was 11 (6 nematodes and 5 cestodes) with 85·6% of the voles carrying at least 1 species and an overall mean species richness of 1·4. At the component community level, the fewest species of helminths were recorded from site 2 (n = 6, compared with 9 at each of the other sites), but site 3 had the lowest Berger-Parker Dominance Index and the highest Simpson's Index of Diversity. At the infracommunity level, site 3 had the highest mean no. of helminth species/vole, the highest mean Brillouin's Index of Diversity but the lowest mean no. of helminths/vole. Voles from sites 1 and 3 differed in the nematodes that were most common (site 1, Heligmosomum mixtum – 95%; site 3, Heligmosomoides glareoli – 79·3%). At site 2 no species exceeded 50% but prevalence of Syphacia petrusewiczi was higher than at the other sites. The prevalence of cestodes was too low to test reliably (12·9%), but the highest prevalence of adult cestodes was recorded at site 1 (22·5% compared with 4·9 and 1·7% for sites 2 and 3 respectively). Host sex did not influence infection, but mean species richness increased with age. The different sites were responsible for most of the variation in our data, and the intrinsic factors (sex and age) were less important in shaping the component community structure of helminths. We conclude that even locations in relative close proximity to one another (13–25 km), selected on the basis of similar habitat quality, have rodent populations that differ in their helminth parasite communities, although for reasons other than the factors quantified in the present study.

The results of the studies on our model combination Trichobilharzia ocellata–Lymnaea stagnalis, presented in this review, lead to the conclusion that schistosomes use multiple strategies to reach their goals, i.e. to propagate and to continue their life cycle. They have to escape from being attacked by the internal defence system (IDS) of the snail host and to profoundly affect the host's energy flow, of which reproduction and growth are the main determinants, for their own benefit. These physiological changes they establish mainly by interfering with the two regulatory systems in the snail host, the IDS and the neuroendocrine system (NES). Moreover, these two regulatory systems clearly interact with each other. Parasitic E/S products affect the host's IDS both in a direct and an indirect way. The neuropeptides or neuropeptide-like substances that are secreted by parasite glands into the host directly suppress haemocyte activity in the snail. The indirect effects include effects of (1) peptides from connective tissue cells and (2) neuropeptides from NES and/or IDS. Parasitic E/S products also induce the effects on energy flow in the host. These E/S products act either directly on a target, as shown for the inhibiting effect of the parasite on the development of the male copulation organ, or on the NES regulating reproductive activity, e.g. on gene expression. Indirect effects of E/S products on the NES (hormone-receptor interaction, electrical activity) are mediated by a factor from connective tissue cells, presumably belonging to the IDS. The physiological changes in the snail host are obviously of vital importance for the parasites, since they make use of different strategies to bring them about.

Voltage-gated calcium (Ca2+) channels contribute to impulse propagation in excitable cells and also regulate intracellular levels of Ca2+. High voltage-activated (HVA) Ca2+ channels are heteromultimeric membrane proteins. The pore-forming, voltage-sensing subunit is the α1 subunit. We have cloned 3 HVA Ca2+ channel α1 subunit cDNAs from Schistosoma mansoni. One of these sequences most closely resembles the L-type class of HVA α1 subunits. The other two sequences are most closely related to non L-type α1 subunits. These schistosome α1 subunits have many of the features common to HVA Ca2+ channels, but also have distinct structural motifs. Analysis of the structural and functional properties of schistosome Ca2+ channel subunits may provide information about these critical components of excitable cells.