Contrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin–trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin–trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to ‘see’ sensitive parasite proteases sufficiently to mount a response against the parasite.

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.

Parasite-induced modifications of host behaviour are known from a wide range of host-parasite associations. In many cases, these behavioural changes are thought to be adaptive and. benefit the parasite by increasing its probability of successful transmission. However, in many cases, energy spent on host manipulation will not be available for other functions, such as growth. These trade-offs suggest that in the absence of other constraints, natural selection will optimize, and not maximize, the influence of parasites on host behaviour. This argument is developed and expanded into theoretical considerations of the evolution of host behaviour manipulation by parasites. Among populations of the same parasite species or among closely-related species, the optimal investment into manipulation, or optimal manipulative effort (ME*), of individual parasites is predicted to increase as (1) typical infrapopulation size decreases, (2) prevalence increases, (3) the longevity of the infected host, or of the parasite in its host, decreases, (4) passive transmission rates decrease, and (5) parasite fecundity decreases. This evolutionary analysis indicates that ecological and life history variables may have played an important role in the evolution of manipulation of host behaviour by parasites.

Factors which affect adhesion of cultured Trypanosoma congolense bloodstream forms to mammalian feeder cells have been examined. Using an in vitro binding assay, the initial events following interaction of trypanosomes with bovine aorta endothelial (BAE) cells were monitored by both light- and electron microscopy. Metabolic inhibitors and other biochemicals were incubated with either cells or parasites, to test whether any inhibited the process. Our findings suggest that adhesion of the parasites is an active process requiring metabolic energy from the trypanosomes, but not from endothelial cells. We also provide data suggesting that T. congolense bloodstream forms possess a lectin-like domain, localized at distinct sites on their flagellar surface, which interacts with specific carbohydrate receptors, most likely sialic acid residues, on the endothelial cell plasma membrane. We also suggest that the cytoskeletal protein actin is probably involved in this interaction.

Two clones of East African Trypanosoma brucei, with distinct homozygous isoenzyme patterns for one of three enzymes examined, were cotransmitted through the tsetse fly vector Glossina morsitans centralis. Flies with mature infections were individually fed on mice and the subsequent bloodstream form populations analysed for the presence of hybrid trypanosomes by isoenzyme analysis. Several combinations have previously been detected using this approach (Schweizer, Tait & Jenni, 1988; Sternberg et al. 1989). Four clones were isolated from one of the hybrid-containing populations. They showed a hybrid phenotype, as would be expected for the F1 progeny in a diploid Mendelian system. The analysis of the progeny clones, using two gene probes which detect restriction fragment length polymorphisms between the two parental stocks, showed that alleles had segregated at each locus and given rise to three different non-parental combinations of alleles in the hybrid progeny. Characterization of the hybrid progeny clones by PFGE (pulsed field gradient gel electrophoresis) revealed that all progeny clones were recombinant for the intermediate size chromosomes. From the analysis of the segregation of the larger chromosomes, marked by P0K (phosphoglycerate kinase) and CP (cysteine protease) gene probes, it was inferred that the progeny clones did not result from a direct fusion of diploid cells. Results with the PGK probe fit into a classical system with meiosis and subsequent fusion of the nuclei to form diploid progeny. On the other hand, blots with the CP probe as well as some of the ethidium bromide stained PFGE gels revealed the existence of non-parental size chromosomes in some of the hybrid progeny. This phenomenon was observed previously (Gibson, 1989) and further investigation is required to elucidate the mechanism.

Sensitive assays capable of detecting proteinases in single females of the phytoparasite Globodera pallida have been developed and used to define the proteinase activity of young adult females. Digestion of the large subunit of the plant protein Rubisco established a pH optimum for the proteinase activity at pH 5·7. The activity was inhibited by the cysteine proteinase inhibitors p-chloromercuribenzoic acid (PMBA) and p-chloromercurisulphonic acid (PMSA) and stimulated by both cysteine and dithiothreitol (DTT). It was moderately reduced by L-trans-epoxysuccinyl-leucylamido-(4- guanidino) butane (E64) but not by specific inhibitors of serine, aspartate or metallo-proteinases. The activity separated into 3 bands on a non-denaturing gel but only I proteinase of 62 kDa was recovered following a combination of anion-exchange chromatography and affinity chromatography using PMBA. The effect of inhibitors was similar to that reported previously for some of the cysteine proteinase activity recovered from Caenorhabditis elegans but is apparently not that for which the corresponding gene has been cloned in this nematode and Haemonchus contortus. The proteinase may have a major role in digestion of dietary protein and so offers an exciting target for future control of this important plant-parasitic nematode.

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

Wetanema sp. is a nematode parasite of the hind gut of the freezing-tolerant orthopteran Hemideina maori (a New Zealand alpine weta). The prevalence and intensity of infection remains high throughout the winter, suggesting that the parasite can survive despite the regular freezing of the host. In the laboratory, Wetanema has survived freezing within its host to temperatures as low as −61 °C, much lower than the supercooling point or lower lethal temperature of the weta. The freezing tolerance of the parasite is therefore much greater than that of its host. Female worms survived lower temperatures better than males and juveniles. Parasites might assist the freezing tolerance of their host if they acted as endogenous ice nucleators. However, there was no relationship between the size of the worm burden and the supercooling point of the host and no significant difference between the supercooling points of infected and uninfected hosts. The freezing and subsequent survival of isolated Wetanema was observed directly on a microscope cold stage. This parasite of a freezing- tolerant host is thus also freezing tolerant. There are few other reports of a parasite surviving freezing within a living host.

This study represents an analysis of trypanosome strains circulating within a confined location over a short period of time during a sleeping sickness epidemic in S.E. Uganda. A large number of Trypanosoma brucei isolates (88) were collected from a variety of hosts (man, cattle, pigs and tsetse) from villages within a 10 km radius and were analysed for variation in isoenzyme patterns, restriction fragment length polymorphism (RFLP) in repetitive DNA sequences and susceptibility to human serum. The human infective stocks form a clearly distinguishable population when compared with other stocks circulating in the domestic cattle reservoir. The data here support the occurrence of genetic exchange between the cattle stocks while an ‘epidemic’ population structure involving limited genetic exchange is a characteristic of the human infective stocks. Furthermore, it is shown that when both RFLP and isoenzyme analysis are carried out most stocks appear to have individual genotypes. Stocks which were formerly grouped as zymodemes are better considered as a collection of distinct individuals.

Free-living infective larvae Strongyloides venezuelensis were cultured in Dulbecco's modified Eagle's medium at 25 and 37 °C, and development to the parasitic stage was evaluated using morphological, protein and antigenicity criteria. Few larvae cultured at 25 °C showed development whereas, in most of the larvae cultured at 37 °C, there appeared characteristic changes such as a bulb-like head and droplets under the cuticle with an increase of body width of the larvae. The results obtained from two-dimensional polyacrylamide gel electrophoresis(2D–PAGE) revealed that the protein spot patterns of the larvae cultured at 25 and 37 °C were differentiated by 17 specific spots. In addition, Western blot analysis combined with 2D–PAGE for reaction with serum obtained from an infected rat revealed that protein spots showing immunodominant antigen at 37 °C were almost the same as those of the larvae recovered from the rats rather than those of the larvae at 25 °C. These results strongly suggested that a temperature shift from 25 to 37 °C has an important role in the development of free-living infective larvae to the parasitic stage of S. venezuelensis. The culture system established in the present study was useful for biological and biochemical studies in the development from/of the free-living to the parasitic stage of Strongyloides species.

Parasitism is defined in various ways as an intimate relationship in which one partner, the parasite, lives on or in another, the host, generally at the expense of the latter. Parasitism commonly results in a unique array of host physiological responses and adaptations. Most studies of the physiological effects of parasitism have focused on the pathological consequence of infection and disease. While many physiological changes contribute to pathogenesis, it is now recognized that parasitic infections at sub-clinical levels also produce physiological effects that either ameliorate or may not contribute to the disease process. Moreover, these physiological changes are often manifested by altered host behaviour. Behavioural studies have enabled an ecological- and evolutionary-oriented evaluation of host responses. In this fashion, physiological effects may be assessed as to whether they affect fitness and confer benefit or harm to one or both of the symbionts involved. We briefly examine how these physiological responses, specifically neural, endocrine, neuromodulatory, and immunomodulatory components, may interact to modify host behaviours. We consider the adaptiveness of these responses and how the behavioural patterns elicited may simultaneously appear adaptive for the parasite as well as the host. In addition, we address how parasite-host physiological and behavioural interactions may be altered during the course of parasitism.

We provide evidence from isoenzyme analysis, hybridization with repetitive DNA probes, behavioural studies and morphometrics that 4 trypanosome isolates from Glossina morsitans submorsitans in The Gambia constitute a new species now named Trypanosoma (Nannomonas) godfreyi. The bloodstream trypomastigotes of T. (N.) godfreyi are relatively small with a mean length of 13·7 μm (range: 9·1–21·8 μm) and a mean width of 1·65 μm (range: 0·65–2·69 μm). There is no free flagellum and the marginal kinetoplast is subterminal to a rounded posterior end; the undulating membrane is usually conspicuous. As with other Nannomonas, T. godfreyi developed in the midgut and proboscis of Glossina and infections matured in 21–28 days in laboratory G. m. morsitans. In The Gambia the normal vertebrate host appears to be the warthog, Phacochoerus aethiopicus, although elsewhere other wild and domestic suids may also be implicated in the life-cycle. T. godfreyi was identified unequivocally using a 380 bp DNA probe specific for a major genomic repeat sequence; its isoenzyme profile distinguished it clearly from T. simiae and three strain groups of T. congolense: savannah, riverine-forest and kilifi.

Strongyloides ratti has a complex life-cycle with two adult generations, one free-living and dioecious and one parasitic and female only. The parasitic females reproduce by parthenogenesis, but it is unclear whether this is mitotic or meiotic in nature. This question has been addressed genetically by analysing the progeny of parasitic females that were heterozygous at an actin locus for evidence of allelic segregation. Such progeny were similarly heterozygous showing that segregation had not occurred. It was therefore concluded that reproduction in the parasitic female of S. ratti is functionally mitotic.

Four-day-old worms of the tapeworms Hymenolepis microstoma, H. diminuta and H. nana and newly excysted H. microstoma were exposed in vitro at 37 °C to immune serum from mice infected for 4–1 2 weeks with H. microstoma. Worms were fixed for electron microscopy after intervals of 5 min to 96 h. Within 10–15 min an homogeneous precipitate occurred between the microtriches of 4-day-old H. microstoma and H. nana, while on some areas of H. microstoma the precipitate extended distal to the microthrix border and contained small vesicles (30 nm in diameter) and shed microtriches. In H. diminuta precipitates were not found until 2 h post-incubation. The thickness of the precipitate and the number of small vesicles and shed microtriches increased with time after incubation. Since a similar precipitate occurred on worms kept in complement-depleted immune serum, antibodies alone may induce immune damage. The precipitate on newly excysted H. microstoma lacked microthrix fragments. After 48 h an extensive precipitate was found protruding from the rostellar glands on some H. microstoma, and within the culture vessel. Antibodies may therefore be complexing with tapeworm secretory products.

A dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

This paper presents a method of estimating the potential global morbidity due to human intestinal nematode infections (Ascaris lumbricoides, Trichuris trichiura and hookworms), based on the observed prevalence of infection. The method relies on the observed relationships between prevalence and intensity of infection, and between worm burden and potential morbidity. This approach is shown to be sensitive to the precision of the original prevalence estimates and, in particular, to the degree of spatial heterogeneity in levels of infection. The estimates presented here indicate that several tens of millions of children are likely to suffer developmental consequences from infection, and suggest that the global disease burden of geohelminthiasis may be significantly greater than was supposed.

Behavioural patterns involved in avoiding, repelling, or removing parasites allow mammalian hosts to defend themselves from an array of parasites that threaten the host's fitness in the natural world. Some examples of behavioural defenses and the presumed target parasites are: grooming to remove ticks, grouping to reduce attack rate of biting flies, fly repelling movements to reduce parasitic flies, and selective grazing to reduce intake of faecal-borne parasites. These behavioural defenses are discussed with regard to effectiveness in controlling the target parasites. Parasites have sometimes evolved behavioural strategies of evading, penetrating or disabling these behavioural defenses. These parasite behavioural strategies, though less studied, are discussed. Also discussed is the possibility that host behavioural patterns that may defend against one parasite may be exploited by a different type of parasite to facilitate its own transmission. The interplay between host defensive strategies, the cost versus the effectiveness of such strategies, and a parasite's evasion or exploitation of such strategies, may be useful in understanding some aspects of host-parasite dynamics in nature.

A method was developed, which enables substances to be injected into the feeding structure (syncytium) established by Heterodera schachtii in roots of Arabidopsis thaliana. The technique was used to study the uptake of nutrients by the feeding nematode. The fluorescent dye lucifer yellow CH (LYCH) and fluorescence-labelled dextrans of different molecular weights were injected into the thin and translucent roots of A. thaliana. Such roots are a feature of this plant and they provide optimal conditions for microinjection. Injected LYCH was taken up by feeding juveniles and adults, indicated by the staining of the alimentary duct and the digestive system. Fluorescent dextrans of 3, 10 and 20 kDa but not of 40 and 70 kDa were ingested, suggesting that molecules of a maximum Stokes radius of 3·2 to 4·4 nm are taken up. It is likely that the feeding tube, forming the interface between the plant cytosol and the nematode's digestive system, is responsible for this size exclusion effect. The injected fluorescent substances were not detected in plant cells adjacent to the syncytium or in the root vascular elements. Injections into parts of roots which were infested by several nematodes revealed that feeding H. schachtii individuals may share one syncytium.