The population biology of the schistosome–vector snail Bulinus truncatus was studied in an irrigation system near Marrakech, Morocco using both genetic and demographic approaches. The population genetic survey was conducted in 4 sites, 2 sites being sampled on 2 separate occasions. Individuals were genotyped at 6 microsatellite loci. No variability was found at 4 loci, and the 2 other loci had less than 4 alleles. The differentiation, both spatial and temporal, among populations was extremely weak. The demographic survey was conducted using 2 capture-mark-recapture analyses in 2 separate sites, the first in 1999 and the second in 2000. The second analysis permitted the estimation of parameters based on recent methodological developments (multisite models). Although these studies provided information on several traits, we report here on dispersal only. Both analyses showed that individual dispersal is of the order of a few hundreds of metres per reproductive life, that is the scale of the whole irrigation area. Both the genetic and demographic studies indicated that this area harbours a single – or no more than a few – populations of B. truncatus. This has implications for our understanding of the coevolutionary process between snails and flukes.

A cDNA clone codifying ribosomal protein L25 was isolated from a Leishmania braziliensis cDNA gene library. The alignment of the amino acid sequence deduced from this gene with other proteins revealed that this protein is related to the L23/25 ribosomal protein family. This is so because this protein shows, in its C-terminal end, the rRNA binding domains characteristic of these proteins and at the N-terminal end the NLS sequence necessary for its entry into the nucleus. Southern blot analysis showed 2 copies of gene L25 per genome arranged in tandem position and pointing in the same direction. Northern blot analysis showed that this gene is transcribed in 2 mRNAs when parasite promastigotes are in the logarithmic phase. In order to analyse the antigenic properties of L. braziliensis RPL25, it was purified as a recombinant protein and ELISA-tested against cutaneous, mucocutaneous and Chagasic sera. The results indicate that the recombinant RPL25 from L. braziliensis presents a non-specific reaction that disqualifies it for the diagnosis of cutaneous leishmaniasis. In contrast, some of the synthetic peptides derived from its sequence may serve as promising tools for the diagnosis of this disease.

The tick Boophilus microplus is a 1-host tick that causes important losses to bovine herds, and protective antigens are being investigated in order to develop vaccines that avoid the use of acaricides. Paramyosins are multi-functional invertebrate muscle proteins, whose roles may include host immunomodulation, and seem to be a prominent candidate in a schistosomiasis vaccine. We report here the cloning, expression and characterization of a B. microplus paramyosin (BmPRM). Sequence analysis of the full length coding sequence cDNA shows high identity to other arthropod paramyosin sequences, and the predicted molecular weight, pI and secondary structure are consistent with a typical paramyosin. Western-blot expression analysis indicates the presence of BmPRM in all tissues and developmental stages tested, but not in saliva. The recombinant protein (rBmPRM) was shown to bind both IgG and collagen. Possible implications of these activities with host evasion mechanisms are discussed.

The time-course of the humoral immune response of female blackflies after a challenge with bacteria, different Onchocerca microfilariae species, bacterial endotoxin and microfilarial extract was investigated. Strong bacteriolytic and growth inhibition activities against the Gram-positive bacterium Micrococcus luteus were induced by all agents. Specific differences were found in activity levels and time-course. Notably the endotoxin lipopolysaccharide (LPS) induced a very early, profound bacteriolytic and antibacterial response, which declined within a day after injection. In contrast, the bacteriolytic activities after Escherichia coli D31 and Onchocerca microfilariae infections were lower, but remained elevated over the observation period of 4 days. The bacteriolytic activity was correlated to a haemolymph protein with a molecular weight of around 14 kDa. Anti-Gram-positive activity in the E. coli infected group appeared within the first 6 h. However, it took 4 days in the microfilarial infected blackflies to reach significant levels. The active agent was identified to be a peptide with a molecular weight of around 4–4.5 kDa. Activity against the Gram-negative bacteria E. coli was detected in blackflies injected with E. coli D31, O. dukei microfilariae and microfilarial extract on days 1 and 4 after injection. The immune response in S. damnosum s.l. naturally infected via a bloodmeal on cattle supported the findings of the experimental infections. Similarities of the immune response kinetics between bacterial and filarial infections suggested that intracellular Wolbachia bacteria, released from microfilariae, could be responsible for the antibacterial response. This is supported by the observation that the induction of an immune response in the Drosophila melanogaster mbn-2 cell line by the filarial extract is blocked by polymyxin B, which forms inactive complexes with bacterial LPS.

A pleomorphic neoplasm (PN) is described from sections of Onchocerca volvulus worms in nodules excised from Cameroonian patients. PN is confined to older, non-fecund, female worms, and those classed as moribund/dead. It is mainly composed of small, roundish, basophilic cells of diverse sizes, often forming a ‘rosette’ pattern around amorphous eosinophilic centres. The cells have a high nuclear/cytoplasmic ratio and up to 2–3 mitoses/high-power field; some become grossly enlarged, highly polymorphic and contain large, irregular blocks of chromatin. The eukaryotic PN cells first appear posteriorly in the pseudocoelom, probably from ovarian cells; they spread anteriorly, invading or compressing the uteri. Ivermectin treatment increased the prevalence PN from 3.7% of 1422 female worms in 637 patients before treatment to 17.5% of 1134 worms in 511 patients after 3 years treatment. Ivermectin at 400–800 μg/kg annually, or at 150 μg/kg or 400–800 μg/kg 3-monthly, over 3 years, did not increase the PN prevalence significantly, as compared with standard doses of 150 μg/kg annually. In other small series of African patients, PN prevalence increased in worms 2, 4, 6 and 10 months after ivermectin treatment; but there was no increase after treatment with amocarzine, albendazole or diethylcarbamazine and suramin. PN may partly account for the increased macrofilaricidal action of ivermectin on female O. volvulus in patients treated for 3 years at 3-monthly intervals.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Vertical migration by infective larvae (L3) of 3 species of nematodes was investigated. Upright truncated agarose cones were used to test upward migration, and comparable truncated cone-shaped agarose hollows were used to test downward migration. Flat agarose plates were control surfaces. When placed at the bases of upright cones, 74% of Ancylostoma caninum L3 migrated up, whereas only 16·5% migrated down the indented cones; this latter value was not significantly different from larval migration on flat plates. Strongyloides stercoralis L3 also migrated upward in significant numbers (80%). These larvae also failed to migrate downward under normal conditions. However, when the bottoms of the indented cones were 3–5 °C warmer than the tops, 75·5% of S. stercoralis L3 migrated down. In contrast, Haemonchus contortus L3 showed no tendency to crawl up or down cones, when compared with normal crawling behaviour on flat plates. Thus, L3 of A. caninum and S. stercoralis exhibited negative geotaxis, crawling against the pull of gravity, while H. contortus did not. The biology of these parasites may explain these behavioural differences.

We screened a population of the brackish water crustacean Gammarus duebeni from the Isle of Cumbrae for the presence of vertically transmitted microsporidia. We compared 2 screening techniques; light microscopy and PCR-based detection using generic 16S rDNA microsporidian primers. Fifty percent of females from this population tested positive for vertically transmitted microsporidia. The PCR screen was 100% efficient in comparison with existing LM based screening. In addition, the PCR screen produced bands of 2 sizes suggesting that more than 1 species of microsporidian was present. Sequencing revealed 2 distinct species of vertically transmitted microsporidia; 33% of females were infected with the feminizer Nosema granulosis and 17% were infected with a new species which we provisionally designate Microsporidium sp. On the basis of sequence information, we developed a discriminatory PCR–RFLP test based on MspI and HaeIII digests. This screen allows rapid detection and discrimination of vertically transmitted microsporidia in natural field populations. We applied the PCR–RFLP screen to a second G. duebeni population from the Isle of Man. This population also hosted these 2 parasite species. In total 45% of females harboured N. granulosis and 10% harboured Microsporidium sp. No dual-infected individuals were found in either population. The occurrence of 2 vertically transmitted parasites within a population has implications for our understanding of parasite–host relationships in the field and we discuss factors affecting the dynamics of parasite–parasite competition and coexistence.

We present a simple unscaled, quantitative framework that addresses the optimum use of resources throughout a host's lifetime based on continuous exposure to parasites (rather than evolutionary, genetically explicit trade-offs). The principal assumptions are that a host's investment of resources in growth increases its survival and reproduction, and that increasing parasite burden reduces survival. The host reproductive value is maximised for a given combination of rates of parasite exposure, host resource acquisition and pathogenicity, which results in an optimum parasite burden (for the host). Generally, results indicate that the optimum resource allocation is to tolerate some parasite infection. The lower the resource acquisition, the lower the proportion of resources that should be devoted to immunity, i.e. the higher the optimum parasite burden. Increases in pathogenicity result in reduced optimum parasite burdens, whereas increases in exposure result in increasing optimum parasite burdens. Simultaneous variation in resource acquisition, pathogenicity and exposure within a community of hosts results in overdispersed parasite burdens, with the degree of heterogeneity decreasing as mean burden increases. The relationships between host condition and parasite burden are complicated, and could potentially confound data analysis. Finally, the value of this approach for explaining epidemiological patterns, immunological processes and the possibilities for further work are discussed.

A cDNA was isolated from an adult male Oesophagostomum dentatum gene library by screening with a male-specific, partial expressed sequence tag (EST) probe identified previously using a differential display technique. The full-length cDNA of 642 bp included 5′ and 3′ untranslated regions of 44 and 121 nucleotides, respectively, and encoded a predicted protein with a putative 18 amino acid signal sequence and a mature polypeptide of 14.7 kDa comprising ∼15% cysteine residues. The amino acid sequence showed similarity with a number of proteins from Caenorhabditis elegans, parasitic nematodes, insects and amphibia, all of which contain a trypsin inhibitor-like cysteine-rich domain. A 3-dimensional structure model constructed for the O. dentatum protein (designated OdmCRP) inferred that it is composed of 2 domains, each with 5 disulfide bonds, which are indicative of the Ascaris family of serine protease inhibitors. These findings indicate that OdmCRP, with 2 structural domains relating to functionally active sites, is a new member of this inhibitor family.

The direct effects of phytohormones (auxin and kinetin) and root diffusates on the surface lipophilicity of the plant parasitic nematodes Globodera rostochiensis and Meloidogyne incognita were investigated. The fluorescent lipid probe AF18 (5-N-(octodecanoyl) aminofluorescein) was used to detect surface changes. Root diffusates increased AF18 uptake by G. rostochiensis while it had no effect on M. incognita. Kinetin and auxin decreased AF18 uptake in G. rostochiensis, while they had the opposite effect on M. incognita. Auxin/kinetin ratio was also found to be important in triggering the surface changes, especially at high concentrations. Whether plant nematodes have auxin and/or kinetin binding proteins is discussed as well as the mechanism behind the surface lipophilicity changes due to root diffusates and phytohormones.

Most, if not all, organisms face attack by natural enemies and will be selected to evolve some form of defence. Resistance may have costs as well as its obvious benefits. These costs may be associated with actual defence or with the maintenance of the defensive machinery irrespective of whether a challenge occurs. In this paper, the evidence for costs of resistance in insect-parasite and insect-parasitoid systems is reviewed, with emphasis on two host-parasitoid systems, based on Drosophila melanogaster and pea aphids as hosts. Data from true insect-parasite systems mainly concern the costs of actual defence; evidence for the costs of standing defences is mostly circumstantial. In pea aphids, the costs of standing defences have so far proved elusive. Resistance amongst clones is not correlated with life-time fecundity, whether measured on good or poor quality plants. Successful defence by a D. melanogaster larva results in a reduction in adult size and fecundity and an increased susceptibility to pupal parasitoids. Costs of standing defences are a reduction in larval competitive ability though these costs only become important when food is limited. It is concluded that costs of resistance can play a pivotal role in the evolutionary and population dynamic interactions between hosts and their parasites.

Iran is an important endemic focus of cystic hydatid disease (CHD) where several species of intermediate host are commonly infected with Echinococcus granulosus. Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Iran and characterized using both DNA (PCR-RFLP of ITS1) and morphological criteria (metacestode rostellar hook dimensions). The sheep and camel strains/genotypes were shown to occur in Iran. The sheep strain was shown to be the most common genotype of E. granulosus affecting sheep, cattle, goats and occasionally camels. The majority of camels were infected with the camel genotype as were 3 of 33 human cases. This is the first time that cases of CHD in humans have been identified in an area where a transmission cycle for the camel genotype exists. In addition, the camel genotype was found to cause infection in both sheep and cattle. Results also demonstrated that both sheep and camel strains can be readily differentiated on the basis of hook morphology alone.

The aim of the study was to investigate the longitudinal changes of exsheathment of ovine and bovine 3rd-stage strongylid larvae in an artificial rumen (RUSITEC) and to compare the results with in vivo data obtained from rumen-fistulated sheep. Infective larvae were incubated in nylon mesh bags in the sheep rumen or the RUSITEC apparatus for periods of 1, 6 and 12 h, respectively. The 12 h exsheathment rates in the rumen and the RUSITEC apparatus (in parentheses) were as follows: Haemonchus contortus: 100% (100%), Ostertagia circumcincta: 100% (76%), O. leptospicularis: 100% (100%), O. ostertagi: 53% (59%), Trichostrongylus axei: 100% (100%), T. colubriformis: 37% (36%), Cooperia curticei: 94% (76%), C. oncophora: 95% (89%), Nematodirus filicollis: 0% (N.D.), N. spathiger: 11% (15%), N. battus: 7% (5%), Oesophagostomum venulosum: 17% (9%), Chabertia ovina: 7% (2%), Dictyocaulus filaria: 1% (N.D.). Larvae of Nematodirus spp. and T. colubriformis showed a quick rise of the exsheathment rate 2 h after transfer into the abomasum. These results confirm that exsheathment generally occurs in the part of the gastrointestinal tract immediately anterior to the habitat of the adult parasite. The overall similar course of exsheathment in both systems indicates that the essential stimuli for exsheathment were generated and maintained under in vitro conditions of the artificial rumen. In both systems, the bicarbonate concentration and the pH reflected a similar status of the H2CO3/HCO buffer system, which is known to provide the essential stimuli for larval exsheathment of the abomasal species. These results give evidence that the RUSITEC system represents a valid system for studying the kinetics of exsheathment of strongylid nematodes under in vitro conditions. For 7 of the species investigated the obtained results represent the first data on larval exsheathment in vivo and in vitro.

Hyperplasia of Paneth and intermediate cells is a recently described component of the response of the small intestine of mice to infection with the nematode Trichinella spiralis. To investigate whether this hyperplasia is parasite specific or represents a generic intestinal response to infection, mice were infected with T. spiralis, Nippostrongylus brasiliensis, Heligmosomoides polygyrus or Schistosoma mansoni and tissue samples taken at various time-points post-infection to determine Paneth and intermediate cell numbers. All infections induced Paneth and intermediate cell hyperplasia, but the patterns of response varied between the parasite species concerned, reflecting differences in their relationships with the host. Increases in the numbers of these cells appeared to correlate with known patterns of T-helper-2 immune responses.

Low doses of the acetylcholinesterase-inhibiting carbamate nematicides disrupt chemoreception in plant-parasitic nematodes. Fluorescein isothiocyanate (FITC)/dextran conjugates up to 12 kDa are taken up from the external medium by certain chemosensory neurons in Caenorhabditis elegans. Similar chemoreceptive neurons of the non-feeding infective stage of Heterodera glycines (soybean cyst nematode) fill with FITC and the nuclei of their cell bodies selectively stain with bisbenzimide. The widely used nematicide aldicarb disrupts the chemoreceptive response of H. glycines with 50% inhibition at very low concentrations (ca 1 pM), some 10−6-fold lower than required to affect locomotion. Similarly, the anthelmintic levamisole had this effect at 1 nM. Peptides selected as mimetics of aldicarb and levamisole also disrupt chemoreception in H. glycines and Globodera pallida at 10−3-fold or lower concentration than required to inhibit locomotion. We propose an uptake pathway for aldicarb, levamisole, peptide mimetics and other soluble molecules by retrograde transport along dendrites of chemoreceptive neurons to the cell bodies and synapses where they act. This may prove to be a general mechanism for the low-dose effects of some nematicides and anthelmintics.

Nine groups of lambs were challenged with 500 drug-resistant (R) Ostertagia circumcincta infective-stage larvae (L3) twice-weekly for 6 weeks; 3 of the groups were also given 500 susceptible (S) L3 and another 3 given 3000 L3 twice-weekly. One week prior to parasite challenge, 1 group of each dose rate was administered ivermectin-capsules (IVM-CRCs). Following challenge, a second group from each dose rate was administered oxfendazole to remove susceptible parasites. The remaining groups received no drug treatment throughout the trial. No significant difference in faecal egg count was observed between the groups. The groups left untreated did not differ significantly in resulting worm burden. However, the percentage of the total L3 challenge recovered as 4th-stage larvae or adult worms demonstrated a density-dependent relationship and declined with increasing challenge. Those groups treated with anthelmintic to remove S worms prior to necropsy also showed density dependency as worm burdens were lower at the highest dose rate. In the groups administered IVM-CRCs, no dose-dependent effect on worm burden was observed. These differences resulted in more worms being present at the highest dose rate in the IVM-CRC-treated animals than in those animals treated orally with anthelmintic. Adult female worms from the IVM-CRC high challenge group were significantly longer and contained more eggs in utero than those in the other high challenge groups. These results demonstrate that where resistance is present, IVM-CRCs can modify the density-dependent regulation of the parasite population, resulting in the acquisition of larger resistant worm burdens than might otherwise be expected. This could accelerate the build-up of resistant worm populations and decrease the time until treatment failure.

Nematode infections are subject to density-dependent effects on their establishment, survivorship and fecundity within a host. These effects act to regulate and stabilize the size of nematode populations. Understanding how these density-dependent effects occur is important to guide the development of control strategies against parasitic nematodes and the diseases that they cause. These density-dependent effects have been hypothesized to result from intraspecific competition between parasites for limited resources or from the action of host immune responses. However, no specific evidence exists to distinguish between these two hypotheses. We find that in nematode (Strongyloides ratti) infections, density-dependent effects on parasite establishment, survivorship and fecundity are mediated by the host immune response. These density-dependent effects are only observed late in primary infections and no density-dependent effects are observed in infections in immuno-compromised animals. We find no evidence for intraspecific competition between parasites in experimental infections over a range of doses that encompasses all that is observed in natural infections. We conclude that density-dependent effects due to the immune response will act to regulate S. ratti infections before competition for space or nutrients within the host gut ever occurs.

The surface coat of the 2nd-stage juveniles (J2) of plant-parasitic nematodes is considered to be involved in interactions with microorganisms in the soil and rhizosphere, as well as with the host plant. Characterization of surface antigens might be important in the development of new nematode control strategies. In this study, polyclonal and monoclonal antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes were tested for their binding to the surface coat and secreted–excreted products of M. javanica. Some of the monoclonal and polyclonal antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Western blot analysis of M. javanica surface coat extracts revealed labelling of several polypeptides with a 48 kDa main band for the polyclonal antibody IACR-PC Mi 373, and a 55 kDa main band for PC Mj E2. Further characterization of the antigens recognized by the polyclonal antibody PC Mj E2, in planta, showed that they were present in the parasitic stages J2 and J3 and that the surface coat was shed during root penetration. The hypodermis of the infective juveniles was labelled by PC Mj E2 and the monoclonal antibody IACR-Misec 3F.4, suggesting that these surface antigens are produced in the hypodermis. Nematode behaviour was affected by all the antibodies that bound to the surface coat of the pre-parasitic J2, and we demonstrated that the movement pattern of the M. javanica J2 was affected by these antibodies. Continuous binding of the antibodies to the M. javanica surface inhibited the infection of Arabidopsis thaliana roots on agar plates.