In order to investigate the mode of action of a new class of schistosomicides, the N-alkylaminoalkanethiosulphuric acids, the ‘outer 35S] 2-[(1-methylpropyl)amino]-1-octanethiosulphuric acid was synthesized. Labelling studies of adult Schistosoma mansoni were performed in infected mice and in in vitro incubations. After a single oral dose of the drug to infected mice, 5 metabolites were detected by thin-layer chromatography in organic extracts of male and female adult schistosomes. In vitro studies showed that the same compounds were present in organic extracts obtained from adult male and female worms. One of these metabolites was identified by mass spectroscopy as being the dimeric disulphide derivative of the parent labelled thiosulphuric acid.

The reduction in worm burden obtained by treatment of Schistosoma mansoni with praziquantel and oxamniquine was greater in mice with heavy infections than in relatively lightly infected animals. The reduction in worm burden achieved by each drug correlated with the size of the pre-treatment worm burden (r2 = 0·82 and 0·81 for praziquantel and oxamniquine, respectively). Intensity of infection did not affect the degree of tegumental damage and drug-induced antigen exposure on worms recovered soon after treatment with praziquantel. However, praziquantel-treated worms from mice with heavy infections had significantly more murine antibody attached to the treated-worm surface than worms from praziquantel-treated lightly infected mice. Heavily infected mice had greater levels of circulating anti-worm antibodies than lighter infected mice. The correlation between infection intensity and cure rates achieved by praziquantel and oxamniquine may thus be a reflection of the higher litres of relevant antibody in heavily infected mice mediating death of drug-treated worms.

Estimates of the time delay between the infective bloodmeal and maturation (incubation or maturation time) for 4 trypanosome stocks (2 Trypanozoon and 2 Trypanosoma congolense) show that maturation time in tsetse is not a parasite species-specific constant. The mean incubation time of a Trypanosoma brucei rhodesiense stock (EATRO 2340 – 18 days) was not significantly different from one T. congolense stock (SIKUDA88 – 15·5 days) but was significantly greater than another (1/148 FLY9 – 12·5 days). There was no significant difference in incubation times between male and female Glossina morsitans morsitans for any of the stocks but in both of the Trypanozoon stocks the proportion of female flies producing mature infections was significantly less than in males. However, estimates of gene frequency, assuming a model in which maturation is controlled by an X-linked recessive allele, gave inconsistent results indicating that maturation cannot be controlled by a single sex-linked gene. Maturation was shown to be a tsetse sex-dependent phenomenon in Trypanozoon but not in T. congolense infections. Incubation time was quite variable even for a single trypanosome stock (e.g. standard deviation of 5 days for one Trypanozoon stock); we discuss how this variability can affect disease transmission, and the interpretation of age-prevalence data.

Vertically transmitted parasites are transmitted from generation to generation of hosts usually via the host's gametes. Owing to gamete size dimorphism, the major transmission route is transovarial and selection (on the parasite) favours strategies which increase the relative frequency of the transmitting (female) host sex. These strategies impose unusual selection pressures on the host, and coevolution between hosts and vertically transmitted parasites has been implicated in speciation, in the evolution of symbiosis, and in the evolution of novel systems of host reproduction and sex determination. We review the evolutionary implications of vertically transmitted parasites in arthropods before focusing on strategies of transmission of a parasitic sex ratio distorter in Gammarus duebeni. The efficiency of parasite transmission to new hosts is a key factor underlying the relationship between vertically transmitted parasites and their hosts. Vertically transmitted parasites must overcome 2 bottlenecks in order to ensure successful infection of future host generations: first, transmission from adult to gamete; and secondly, transmission to the germ-line of the infected host. We investigate these 2 processes with regard to transovarial transmission by a microsporidian parasite in Gammarus duebeni. Parasite transmission from adult to eggs is highly efficient, with 96% of eggs of infected mothers inheriting the infection, whereas transmission to germ-line within infected embryos is relatively inefficient (72%). We measure parasite distribution between cells of developing embryos, and use these distributions to infer possible mechanisms of parasite transmission to germ-line. Parasite distribution within the embryo is dependent on host cell lineage, and is not consistent with unbiased segregation between daughter cells. These results indicate that parasites segregate together at host cell division, and may reflect a strategy of differential segregation to the host germ-line. We consider alternative parasite strategies at the cell-level in terms of their evolutionary implications.

Cyclosporin A (CsA) acts as a powerful immunosuppressant through its binding to the cytosolic isomerase, cyclophilin (CyP), forming a complex which inhibits the phosphatase activity of calcineurin. The drug is also selectively anti-parasitic but its mode of action remains unknown. The mouse tapeworm, Hymenolepis microstoma is sensitive to CsA, but the rat tapeworm, H. diminuta is not susceptible either in rats, mice or in vitro. Using these two tapeworm models, the uptake and binding of CsA were examined in relation to parasite cyclophilins. Uptake and compartmentalization of the drug were markedly different in the two species: H. microstoma takes up more drug than does H. diminuta and sequesters more drug into intracellular compartments. Characterization of cyclophilins using both CsA binding and isomerase activity assays reveals that H. microstoma possesses two cyclophilin isoforms (Mr 17700 and 21400) with isomerase activity that is inhibited by CsA. Using identical assays, we have been unable to demonstrate CsA-binding proteins or CsA-sensitive isomerase activity in H. diminuta. These data suggest that the anthelmintic action of CsA relates in some way to the presence and function of parasite cyclophilins.

Little is known about the regulation and control of Schistosoma mansoni gene expression. In order to study such mechanisms a gene reporter expression vector construction, under the control of a promoter region derived from the S. mansoni calreticulin gene was used to transfect the human Jurkat T cell line. The promoter region contains potential TATA and CAAT boxes as well as an AP-1 core element. We show here that transcriptional factors of eucaryotic cells may induce a gene reporter activity under the control of a S. mansoni promoter region. Treatment of stably transfected cells with N-acetyl cysteine (NAC), a well-characterized antioxidant which counteracts the effects of reactive oxygen intermediates, enhanced the AP-1 dependent transactivation. This effect was abolished when the SmCaR promoter region was deleted in the AP-1 site. Electrophoretic Mobility Shift Assays showed that the AP-1 sequence of S. mansoni bound to both S. mansoni extracts and in nuclear extracts from Jurkat cells, thus explaining possible activation of AP-1 by NAC. Finally treatment of S. mansoni schistosomula and adult worms with NAC induced an increased synthesis of calreticulin protein suggesting a possible role of redox mechanisms in the regulation of a calreticulin gene transcription process in S. mansoni.

This study examines the ability of an assay which measures the amount of a schistosome specific antigen (CAA) in the host circulation to reliably reflect relative worm burden. Mice were infected with 5 species of schistosome with a range of infection dose. The levels of serum CAA increased during schistosome maturation. In all species tested CAA levels correlated well with adult worm burden once the parasites achieved sexual maturity and remained relatively stable during the establishment of egg production. The amount of CAA produced varied between species but within each species CAA levels were proportional to worm numbers: no density-dependent effects on CAA levels were observed even when mice carried worm burdens that were very large relative to host size. T-cell deprivation of the host had no effect on the CAA/worm burden relationship in either Schistosoma mansoni or S. haematobium infections and the CAA equilibrium was unaltered in intact mice when reduction of worm fecundity occurred. These data support the use of the CAA as an accurate and robust estimate of relative schistosome burden in man.

Glycans at the surface of adult Schistosoma mansoni were investigated with gold-labelled lectins. The fragile complex of the glycans with the outer membranes could be preserved for electron microscopy by avoiding extensive pre-fixation with aldehydes and by introducing osmium-ferrocyanide as a membrane fixative. Male and female worms were entirely covered with glycans that intensely bound lectins from Erythrina cristagalli and Datura stramonium, suggesting that galactose(β1–4)N-acetylglucosamine residues occur in high numbers in the surface glycans. Similar staining was obtained with lectins from Triticum vulgaris, Glycine max and Ricinus communis agglutinin I, which react with N-acetylglucosamine or terminal galactose residues and bind non-selectively with high affinity to N-acetyllactosamine. Fucose, N-acetylgalactose and sialic acid were not detected with lectins and sialidase treatment. The tegument contained an abundance of glycans with the same lectin reactivities as the surface-expressed molecules, indicating that the worms synthesize and replenish their surface glycans and do not merely adsorb host substances. Glycomimesis is discussed as a mechanism of immune evasion in view of N-acetyllactosamine being a common and weakly immunogenic component in glycans of vertebrate hosts. S. mansoni might disguise themselves with the glycans against attack by immune effectors.

The monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

Eighty-seven of 175 laboratory raised F1 generation brook charr (Salvelinus fontinalis) were resistant to experimental Cryptobia salmositica infection. Susceptibility to the pathogen was not related to age nor to route of infection. Plasma of resistant charr lysed the parasite under in vitro conditions while those of susceptible fish did not. Sperm from 4 susceptible and 4 resistant male charr, from 8 F1 families, were used to fertilize eggs from 2 hatchery raised susceptible charr. Susceptibility of progenies (F2 families) was determined 4 weeks after inoculation of the parasite. All progenies (seven F2 families) of 4 susceptible males were susceptible to experimental infection and their plasma did not lyse the parasite. It was presumed that these susceptible progenies were homozygous recessive (rr), and that the resistant allele was dominant. All progenies (two F2 families) of 1 resistant male (presumed RR) and 2 susceptible females were all resistant to infection and their plasma lysed the parasite. The ratio of resistant to susceptible charr in 6 other F2 families from the other 3 resistant males (presumed Rr) and two susceptible females was about 1:1. The results suggest a single Mendelian locus which determines innate resistance to C. salmositica infection in brook charr and that it is possible to breed Cryptobia-resistant fish.

We have previously identified in membranes of the locomotory muscle of Ascaris suum a phosphoramidon-sensitive endopeptidase which hydrolyses the neuropeptide AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2) by cleavage of the Glu3-Phe4 bond (Sajid & Isaac, 1994). We have determined the properties of this neuropeptide-degrading enzyme of A. suum muscle using AKH-I (ρGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) and [D-Ala2, Leu5]enkephalin as convenient endopeptidase substrates. Phosphoramidon, thiorphan and SQ 28603, potent inhibitors of mammalian neprilysin (neutral endopeptidase, endopeptidase 24.11), inhibited the endopeptidase activity towards AKH-I with IC50 values of 0·13 μM, 22 μM and 6·3 μM, respectively. Two other neprilysin inhibitors (SCH 32615 and SCH 39370) and the bivalent metal ion chelators, EDTA (1 mM) and 1, 10 bis-phenanthroline (1 mM) failed to inhibit the nematode enzyme. The endopeptidase had a neutral pH optimum and a significant proportion (45%)of the enzyme activity partitioned into the detergent-rich phase of Triton X-114, indicating that the enzyme is an integral membrane protein. The muscle enzyme also attacked [D-Ala2, Leu5]enkephalin cleaving the Gly3-Phe4 bond and this hydrolytic activity was inhibited by phosphoramidon and thiorphan (IC50, 0·28 ρM and 15·8 ρM, respectively) but not by EDTA and 1,10 bis-phenanthroline. The phosphoramidon-sensitive endopeptidase activity was detected on intact muscle cells prepared by collagenase treatment of the body wall musculature, indicating that endopeptidase is accessible to peptide molecules that interact with the cell surface.

Seven cDN A clones of Schistosoma mansoni containing the C-terminal part of the deduced sequence of a mucin-like protein have been identified. The protein contains 28% threonines, 20% serines, and has a pi of 3middot;4. On Northern blots of RNA of adult worms, the cDNA clones detect 2 transcripts of 1middot;65 and 4middot;2 kb which are expressed only in female worms. The tissue of gene expression, as revealed by in situ hybridization, is the epithelium surrounding the female reproduction duct proximal to its entrance into the ootype. Accumulation of N-glycosylation sites suggests that the protein, like other mucins, might form a protective layer, coating the lining of the duct. Regarding its acidic pi, we hypothesize a role in preventing premature egg-shell formation. This is the first female-specifically transcribed sequence, hitherto known in S. mansonithat is not expressed in the vitellaria.

In this paper we review the published literature on patterns of abundance and aggregation of macroparasites in wildlife host populations. We base this survey on quantitative analyses of mean burden and a number of measures of the degree of aggregation of parasite burdens between hosts. All major parasite and vertebrate host taxa were represented in the database. Mean parasite burden was found to be log-normally distributed, indicating that all parasite burdens are regulated to some degree. In addition, all but one of the parasitic infections were aggregated with respect to their hosts, and the relationship between log mean parasite burden and log variance was found to be very strong (R2 = 0·87). That is, for a given mean parasite burden there are constraints on the degree of variation in individual host burdens. The aggregated nature of the parasitic infections is also apparent from other measures of the degree of aggregation: prevalence – mean relationships, and the negative binomial parameter, k. Using a relatively new technique for parasitological infection data – tree-based models, as well as traditional linear models – a number of the parasitic infections was found to be associated with systematically lower or higher parasite burdens. Possible biological explanations for these and other patterns are proposed.

Monoclonal antibody 2A6 binds to the surface of the Dictyocaulus viviparus L3 sheath and identifies a molecule which is highly antigenic in both infected and vaccinated cattle. Immunogold electron microscopy reveals that the antibody binds to a highly stable surface coat overlying the epicuticle of the L3 sheath. The binding to the cuticular surface is stage specific, being restricted to the surface of the L3 sheath, although the antigen can be detected on Western blots of parasite homogenates from the L1 stage. Immunofluorescence on live parasites demonstrates that the antibodies also bind to the L3 sheath surface of many other strongylid nematodes. The antigen detected by 2A6 differs in molecular weight between the species; in D. viviparus the antigen is poorly resolved between 29 and 40 kDa by SDS-PAGE whereas in nematodes from the family Trichostrongyloidea a single band or doublet of approximately 20 kDa is detected, suggesting that a related molecule is present in these species. Although infection and vaccination with D. viviparus elicits a strong immune response to the L3 sheath antigen this does not appear to be the case following infection with other strongylid nematodes.

The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region. The nucleotide sequences reported in this paper have been submitted to the EMBL Data Library under accession numbers Z26942 (G. salaris), Z35128 (G. derjavini) and Z35129 (G. truttae).

The evolution of faecal egg output, worm burdens and tissue egg counts in young calves was monitored during the first year of natural exposure to Schistosoma mattheei infection on a Zambian farm. According to the duration of their stay on the farm, these calves were classified into 2 groups of 14 temporary tracers (TT calves) which were introduced on a 2-monthly basis for residential periods of 2 months, and 12 permanent tracers (PT calves) introduced either at the beginning of the experiment (Group A) or 2 months later (Group B) and gradually removed after residential periods of 2, 4, 6, 8, 10 and 12 months on the farm. Worm counts in the TT calves showed that infection occurred throughout the year on the farm and that levels of infection acquired during each period of 8 weeks correlated well with the respective infected snail densities observed at the main transmission site. Marked differences in worm population dynamics were recorded between the 2 groups of PT calves. In Group B animals which apparently were initially exposed to heavy transmission, according to the results from TT calves, much higher worm counts and greater susceptibility to reinfection were observed than in Group A animals initially exposed to lighter exposure. These results suggest that the development of resistance to natural infection with S. mattheei may depend on the initial exposure to the parasite. Low initial exposures may lead to resistance whereas high initial exposures may result in decreased immune responses resulting in susceptibility to infection.

The taxonomic history of members of the 37-collar-spine group within the genus Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5·8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cort, 1914), E. revolution (Frölich, 1802), E. caproni Richard, 1964, E. liei Jeyarasasingam et al. 1972, E. paraensei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. hortense (Asada, 1926). Five of the eight species were clearly distinguishable using ITS data. Sequences from the remaining three taxa, E. caproni, E. sp.II and E. liei were identical to one another and the group containing these taxa was distant from other 37-collar-spine species on a phylogenetic tree. E. trivolvis and E. paraensei form a second, but less distinct group within the 37-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

Adult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14–100 kDa were observed with optimal activity at pH 7·5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.

Syngamus trachea, the gape nematode, has been used as a model to study the chemosensory function of amphids. Extracellular electrophysiological recordings were performed directly on amphids. The amphids were stimulated by blood serum from a host bird and by D-tryptophan. The spike frequency produced by the amphid increased significantly after the application of the serum and the application of D-tryptophan. Two types of amphidial neurones responded to the presence of the stimuli. These extracellular recordings have verified the chemosensory function of the amphids.

The characteristically aggregated frequency distribution of macroparasites in their hosts is a key feature of host–parasite population biology. We begin with a brief review of the theoretical literature concerning parasite aggregation. Though this work has illustrated much about both the sources and impact of parasite aggregation, there is still no definitive analysis of both these aspects. We then go on to illustrate the use of one approach to this problem – the construction of Moment Closure Equations (MCEs), which can be used to represent both the mean and second moments (variances and covariances) of the distribution of different parasite stages and phenomenological measures of host immunity. We apply these models to one of the best documented interactions involving free-living animal hosts – the interaction between trichostrongylid nematodes and ruminants. The analysis compares patterns of variability in experimental infections of Teladorsagia circumcincta in sheep with the equivalent wildlife situation – the epidemiology of T. circumcincta in a feral population of Soay sheep on St Kilda, Outer Hebrides. We focus on the relationship between mean parasite load and aggregation (inversely measured by the negative binomial parameter, k) for cohorts of hosts. The analysis and empirical data indicate that k tracks the increase and subsequent decline in the mean burden with host age. We discuss this result in terms of the degree of heterogeneity in the impact of host immunity or parasite-induced mortality required to shorten the tail of the parasite distribution (and therefore increase k) in older animals. The model is also used to analyse the relationship between estimated worm and egg counts (since only the latter are often available for wildlife hosts). Finally, we use these results to review directions for future work on the nature and impact of parasite aggregation.