New biological species and high levels of inter- and intraspecific genetic divergence were discovered in an allozyme study of some North European members of the acanthocephalan genus Echinorhynchus (sensu lato), parasites of fish and malacostracan crustaceans. (i) A strong differentiation between the marine E. gadi and the fresh- and brackish-water E. salmonis (genetic identity I ≃ 0) supports a generic distinction between these taxa; however, the subdivision would not entirely concur with the concepts of Echinorhynchus (sensu stricto) and Metechinorhynchus suggested earlier. (ii) Samples of E. gadi from the Baltic, Norwegian and North Seas included three distinct, partially sympatric biological species (spp. I–III; I ≃ 0·5). (iii) E. bothniensis, previously only known from the northern Baltic Sea, represents a complex of freshwater taxa with an intermediate host relationship to the ‘glacial relict’ Mysis spp. and with a distributional and host analogy to the North American E. leidyi. A population in a northern lake in the Barents Sea basin is closely related to E. bothniensis of the Baltic area, but is probably specifically distinct; the divergence between these populations (I ≃ 0·6) is similar to that between their Mysis host species. (iv) Considerable intraspecific differentiation (Fst = 0·25), probably reflecting post-glacial population bottlenecks, was found between Baltic and nearby lacustrine E. bothniensis, and between Atlantic and Baltic E. gadi sp. I.

After demonstrating that rats were capable of discriminating between the same diet treated with either flavour 1 or flavour 2 and that the 2 flavours of diet were equipreferred, an experiment was carried out to see whether learned taste aversion might play a role in the reduction of food intake that is commonly observed during the course of a parasitic infection. The results showed that rats, given a subcutaneous inoculation of approximately 6000 third-stage larvae of Nippostrongylus brasiliensis (Nematoda) while feeding on a diet containing flavour 2, strongly preferred to eat diet containing flavour 1 when given simultaneous choice conditions. Uninfected rats showed no preference and ate equal amounts of both flavoured diets. This effect is interpreted as the first experimental demonstration of learned taste aversion using a eukaryotic parasite as the inductive agent.

Artificially transformed schistosomula of Schistosoma mansoni develop a consistent but small protection against the lethal effects of antibody plus complement when cultured for 24 h in a chemically defined medium. In contrast, they become rapidly resistant to antibody plus complement, when cultured in the presence of a complex medium consisting of equal parts of heat-inactivated rabbit serum and Earle's/lactalbumin or in defined medium supplemented with small amounts of heat-inactivated rabbit serum. Sephadex G-200 gel filtration revealed that the protective factor in rabbit serum is a macromolecule with a molecular weight between 7 and 19S. Parasites cultured at 10 °C or in the presence of 200 μg of puromycin show less serum-induced protection against the lethal effects of antibody plus complement than do controls.

The eggs of D. coeliaca have been cultured successfully and the free-swimming larva has been studied for the first time. The eggs have an incubation period which for monogeneans is exceptionally long (4–5 months at 10 °C). Apart from maintenance at a low temperature no other special conditions such as high hydrostatic pressure or washing to remove host body fluid are necessary for development. It is not necessary for the eggs to come into contact with host body fluid before development can begin.

The oncomiracidium of D. coeliaca has no eyes, a well-developed pair of anterior median gland cells and a pair of hamuli. The oncomiracidium of C. kroyeri is similar to that of D. coeliaca except for the presence of two pairs of conspicuous pigmented eyes, poorly developed anterior median gland cells, the presence of an extra pair of gland cells at the posterior end of the body and the absence of hamuli.

A comparison of the larval features of D. coeliaca and C. kroyeri has confirmed that these parasites belong to quite distinct species.

The biology and life cycle of Apatemon gracilis minor is briefly described.

The morphology of the cup–shaped forebody with the small lappets, the pear-shaped subcuticular cells and the very flexible finger–like adhesive organ lobes is considered in some detail.

An extensive description of the body musculature is given and the mode of attachment of the parasite to the host mucosa is studied.

The main results of the histochemical tests are: (a) a relative absence of enzymes in the adhesive organ and a greater activity in the lappets; (b) the presence of two types of esterase, a non-specific esterase of B-type in the caecal cells and a non-specific esterase of resistant type, possibly a non-specific cholinesterase, in the rest of the reacting sites in the parasite; (c) a relatively small activity for leucine aminopeptidase; (d) the presence of RNA at all sites of enzyme formation; and finally, (e), the presence of enzymes and carbohydrates in the cuticle.

The activities of the parasite result in a local disruption of the host intestinal mucosa at the site of attachment. It is suggested that this is a result of the activities of the enzymes secreted by the parasite.

The absorption of nutrients and the circulatory function of the excretory system are discussed.

I should like to express my sincere thanks to Dr D. A. Erasmus for helpful discussions and advice during the progress of the work and to Professor J. Brough for giving me the opportunity to work in his department. The study was conducted during the tenure of a grant from Svenska Vetenskapliga Centralrådet i Finland.

Scanning and transmission electron microscopy of Trypanosoma congolense epimastigotes attached to a plastic substratum shows them to elaborate a complex flagellum filament system and plaque with a highly organized structure. Non-ionic detergent extraction of these cells shows that the resulting cytoskeletons remain attached to the plaque. The subpellicular corset of microtubules can be removed by salt or Ca+2 treatment leaving the axoneme, paraflagellar rod, associated filaments and the plaque. Neither of these treatments therefore removed the plaque-associated material from the substratum. Analysis of these fractions by SDS–polyacrylamide gel electrophoresis reveals an abundant 70 kDa protein that is highly enriched in the salt extracted ‘minimal plaque’ structures and appears likely to be a major constituent of this structure. These studies reveal that the complex filament and microtubule systems of the cytoskeleton involved the attachment of trypanosomes to substrata and have established a method of biochemical fractionation of the structures and components involved.

The understanding of the mechanisms whereby intracellular parasites counteract the microbicidal processes of macrophages has progressed considerably in recent years. Various factors contribute to intracellular parasite destruction; from a biochemical standpoint, particularly important is the oxidative burst triggered by phagocytosis and by macrophage ‘activation’, that leads to the generation of toxic metabolites of oxygen. At the ultrastructural level, fusion of the parasitophorous vacuole with surrounding lysosomes appears to be a pre-requisite for the final digestion and elimination of the infecting microorganisms. The counter-measures evolved by microorganisms to escape intracellular destruction are best illustrated by studies in vitro on the interaction of parasites of the Leishmania, Toxoplasma and Trypanosoma spp. with mononuclear phagocytes. Some microbes are able to inhibit the fusion of phagosomes with lysosomes, thus avoiding the potentially harmful action of lysosomal hydrolases. Other microorganisms are able to resist the effects of such enzymes, perhaps by secreting inhibitory substances. Others still avoid lysosomes by leaving the phagocytic vacuole, to reach the cytoplasmic matrix where their development is unhindered. Particularly critical is the capacity of certain parasites to subvert the lethal effects of the oxidative burst. This can be achieved either by failing to evoke this metabolic response, or by producing scavengers that can detoxify harmful oxygen metabolites. Intracellular death or survival will thus depend on a delicate balance between the potency of macrophage cidal mechanisms, and the efficacy of the protective measures evolved by the infecting agents.

Trypanosomes were studied from the blood of 6 species of small British fish caught in the River Lee. Morphologically the trypanosomes from the blood of Nemacheilus barbatulus L., Phoxinus phoxinus L., Cottus gobio L., Gobio gobio L., Gasterosteus aculeatus and Pungitus pungitus L. were indistinguishable. Cross-transmission experiments using syringe passage of culture forms and also the leech vector Hemiclepsis marginata showed that the trypanosomes were not host specific. The isoenzyme patterns of culture forms from N. barbatulus and P. phoxinus were identical for 11 enzymes studied. The trypanosomes from the 6 species of fish previously classified as separate species are, on the basis of these results, regarded as a single species, Trypanosoma cobitis (Mitrophanow, 1883).

We present a discrete time model for age-related changes in the mean and variance of the number of helminth parasites per host. We assess the degree of aggregation as the negative binomial parameter, k, and use the model to examine the influence of various factors on changes in aggregation with host age: discrete versus continuous infection; the degree of predisposition to infection; infection rate; parasite survival rate; and the variance in exposure to infective stages. The model can produce both increases and decreases in k with host age. However, with parameter values typical of many human helminth infections, a monotonic increase in k (decrease in aggregation) with age is predicted. With an age-dependent infection rate, convex relationships between k and age are possible. These predictions are consistent with data from field studies, but differ from those of previous models which have suggested that k is independent of host age in the absence of density dependence in parasite population dynamics. Differences between the models, and some difficulties in the interpretation of field data, are discussed.

The European shore crab, Carcinus maenas, is one of the most successful marine invasive species. Its success has been in part attributed to the loss of parasites, rekindling an interest in host-parasite interactions and impacts on host fitness in this crab. In the present study, we investigated C. maenas populations from Europe, South Africa and Australia for parasites, and assessed their impact on the fitness of male crabs. For the shore crab, testes weight along with success in mating competition is traded off against other life-history traits. We therefore used this parameter as an indicator both for reproductive fitness and a possible resource trade-off in response to parasite infestation. In the native range, crabs infested with Sacculina carcini showed significantly lower testes weight than uninfected crabs. However, helminth parasites did not generally cause reduced testes weights. Crab populations from South Africa and Australia were either parasitized at very low prevalences, or were completely parasite free. However, no population level effect of this parasite release was reflected in testes weight. These findings do not support a severe fitness impact of helminth parasites on C. maenas, which questions the role of parasites on its population dynamics, both in the native area and for invasive success.

Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of β-tubulin gene). Six flocks (F1−F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for β-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.

In parasitological research, significant progress has been made with respect to genomics and transcriptomics but transgenic systems for functional gene analyses are mainly restricted to the protozoan field. Gene insertion and knockout strategies can be applied to parasitic protozoa as well as gene silencing by RNA interference (RNAi). By contrast, research on parasitic helminthes still lags behind. Along with the major advances in genome and transcriptome analyses e.g. for schistosomes, methods for the functional characterization of genes of interest are still in their initial phase and have to be elaborated now, at the beginning of the post-genomic era. In this review we will summarize attempts made in the last decade regarding the establishment of protocols to transiently and stably transform or transfect schistosomes. Besides approaches using particle bombardment, electroporation or virus-based infection strateies to introduce DNA constructs into adult and larval schistosome stages to express reporter genes, first approaches have also been made in establishing protocols based on soaking, lipofection, and/or electroporation for RNA interference to silence gene activity. Although in these cases remarkable progress can be seen, the schistosome community eagerly awaits major breakthroughs especially with respect to stable transformation, but also for silencing or knock-down strategies for every schistosome gene of interest.

One of the most common causes of calf diarrhoea is the parasite Cryptosporidium parvum. Two longitudinal studies were carried out on a dairy farm Scotland to determine the prevalence of Cryptosporidium species and subtypes in a group of calves and to determine whether dams were a possible source of calfhood infection. Fecal samples were collected from 25 calves from birth to 12 months in the first year. In the second year, fecal samples were collected from pregnant cows (n = 29) and their calves (n = 30) from birth to 6 months. The samples were tested for Cryptosporidium and speciated. Cryptosporidium parvum-positive samples were subtyped by GP60 fragment analysis. All calves in both studies shed Cryptosporidium during the study period. Cryptosporidium parvum was the predominant species detected in calves ⩽6 weeks of age and at 6 months of age, C. bovis and C. ryanae were detected in calves older than 4 weeks of age but ⩽6 months of age. The prevalence of Cryptosporidium was higher in younger animals than in older animals. GP60 subtyping revealed two subtypes in calves on this farm (IIaA15G2R1 and IIaA19G2R1) that differed in frequency by age. Adult cattle also shed C. parvum, of four gp60 genotypes.

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.

A quick and simple method of purifying the histones from Plasmodium falciparum culture supernatant or from infected erythrocytes is described. The proteins were present only in preparations rich in P. falciparum nuclear material and were soluble at acid pH and in strongly anionic detergents. Four proteins, of 14–18 kDa were identified as the P. falciparum core histones. N-terminal sequence analysis of the 16 and 18 kDa proteins and of a tryptic fragment of the protein mixture revealed strong homologies with deduced cDNA or protein sequences of histones H2A, H2B, and H3 of P. falciparum and other species. Antibodies raised against the proteins cross-reacted weakly with histones of other species and, on immunofluorescence, localized the proteins to schizont nuclei. Anti-P. falciparum histone antibodies were detected in sera of semi-immune human adults but these antibodies did not react with human histones on a Western blot. The large quantities of P. falciparum histone released and the chronic nature of malarial infection, together with the unusually high avidity of histones for ligands found in renal and vascular basement membrane, raise the question of a role for histones in the pathogenesis of malarial infection. Wesuggest that histones or histone±antibody complexes may contribute to disease pathology.

We examined the effects of Haemoproteus infection on the survival and pathology caused in the biting midges. Forty-six females of Culicoides impunctatus were exposed experimentally by allowing them to feed on a naturally infected red-backed shrike infected with Haemoproteus lanii (lineage hRB1, gametocytaemia 5·2%). Seventeen females were fed on an uninfected bird (controls). Dead insects were collected, counted and used for dissection, histological examination and polymerase chain reaction-based testing. Parasites were present in all experimentally infected biting midges, but absent from control insects. Survivorship differed significantly between the control and infected groups. Twelve hours post-exposure (PE), 45 (98%) experimentally infected midges were dead, but all control midges remained alive, and many of them survived until 7 day PE. The migrating ookinetes of H. lanii overfilled midgut, markedly damaged the midgut wall, entered the haemocoel and overfilled the abdomen and thorax of exposed biting midges. Massive infection by migrating ookinetes led to damage of abdomen and thorax of biting midges. The parasites often present in large clumps in the haemocoel in abdomen and thorax, leading to the interruption of the haemolymph circulation. These are the main reasons for rapid death of biting midges after feeding on high-intensity infections of Haemoproteus parasites.

The co-occurrence of different pathogen species and their simultaneous infection of hosts are common, and may affect host health outcomes. Co-infecting pathogens may interact synergistically (harming the host more) or antagonistically (harming the host less) compared with single infections. Here we have tested associations of infections and their co-infections with variation in growth rate using a subset of 455 animals of the Infectious Diseases of East Africa Livestock (IDEAL) cohort study surviving to one year. Data on live body weight, infections with helminth parasites and haemoparasites were collected every 5 weeks during the first year of life. Growth of zebu cattle during the first year of life was best described by a linear growth function. A large variation in daily weight gain with a range of 0·03–0·34 kg, and a mean of 0·135 kg (0·124, 0·146; 95% CI) was observed. After controlling for other significant covariates in mixed effects statistical models, the results revealed synergistic interactions (lower growth rates) with Theileria parva and Anaplasma marginale co-infections, and antagonistic interactions (relatively higher growth rates) with T. parva and Theileria mutans co-infections, compared with infections with T. parva only. Additionally, helminth infections can have a strong negative effect on the growth rates but this is burden-dependent, accounting for up to 30% decrease in growth rate in heavily infected animals. These findings present evidence of pathogen–pathogen interactions affecting host growth, and we discuss possible mechanisms that may explain observed directions of interactions as well as possible modifications to disease control strategies when co-infections are present.

The recombinant Echinococcus multilocularis antigen ll/3–10 is one of the most promising tools for immunodiagnosis of alveolar echinococcosis in human patients. Its nucleic acid sequence represents a part of the E. multilocularis gene encoding the metacestode antigen II/3, the former being basically present and expressed in both E. multilocularis and E. granulosus. Most (94%) patients with alveolar echinococcosis respond to infection with a marked anti-II/3–10 IgG synthesis; in contrast, most of the cystic echinococcosis patients do not, for some reason, recognize the recombinant antigen. We tackled this problem by generating cDNA derived from both E. granulosus and E. multilocularis full length II/3 genes, performed by reverse transcription and PCR amplification. Sequence analysis revealed a very high degree of conservation of the primary sequence of the antigen II/3 in both Echinococcus species. cDNA fragments were subcloned and expressed in E. coli as fusion proteins with Schistosoma japonicum glutathione S-transferase. Recombinant proteins were affinity purified and comparatively assessed by ELISA with respect to antibody-binding characteristics. Sera from patients suffering from cystic echinococcosis showed no significant differences in reactivity with the antigens derived from either E. multilocularis or E. granulosus. Therefore, parameters other than some minor differences in the primary sequence seem to be responsible for the lack of antigen II/3 recognition in cystic echinococcosis.

Note

Nucleotide sequence data reported in this paper have been submitted to the GenBank® data base with the accession numbers U05573 and U05574.

Density-dependent effects on adult and larval stages of the introduced nematode parasite Anguillicola crassus in the European eel definitive host were examined in the laboratory in naturally infected fish, and in field samples. The effect of adult nematode subpopulations on larval development over time and the effect of increasing adult intensity on the gravid female subpopulations were investigated. At high adult subpopulations the movement of larvae from the swimbladder wall into the swimbladder lumen appears to be inhibited, and A. crassus larvae to be arrested in development in a density-dependent manner. The number of gravid females per host reaches a constant level, and so the proportion of gravid female nematodes per adult subpopulation decreases relative to further increasing total adult nematode numbers. Both these mechanisms have the potential to regulate infrapopulations of A. crassus within the eel definitive host and thus the parasite suprapopulation.

Substrate preferences, spatial aggregation patterns and seasonal dynamics in the egg laying of ectoparasitic Argulus coregoni were studied at a commercial fish farm in Finland. Pilot experiments showed that A. coregoni females selected specific types of substrates for egg laying. Significantly more A. coregoni eggs were laid on dark substrates than on light ones suggesting the use of visual cues. Therefore, egg-laying plates of dark colour were constructed for further experiments. Most A. coregoni eggs were deposited in locations in shadow and in the deepest water in a 2 m deep farming canal. Relatively more eggs were laid on bottom stones situated near each egg-laying trap than on artificial egg-laying plates indicating a preference for irregular stones in the deeper locations in the canal. The plates were located 20 cm above the bottom. However, a total of 5863 A. coregoni egg clutches, corresponding approximately to 1·5 million unhatched metanauplii, were successfully destroyed with the plates indicating that egg-laying traps can be used as an ecological control method against argulids in certain situations. For traps to be effective, ponds should not contain stones or any other hard substrata attracting female lice. The egg laying of A. coregoni in this study started on 5 July in 2001 and extended over 3·5 months up to mid-October. The egg-laying pattern of A. coregoni population was unimodal, supporting the view that only a single A. coregoni generation occurred annually in Central Finland.