The uptake kinetics of a 14C-labelled trypanocidal compound isometamidium chloride (SamorinR, RMB Animal Health Ltd, UK) was measured in drug-resistant and drug-sensitive Trypanosoma congolense. It was established that drug uptake was significantly more rapid and quantitatively greater in drug-sensitive parasites. There was clear evidence that drug uptake in both the resistant and sensitive trypanosomes was by a specific, receptor-mediated process. This specific drug transport was energy-dependent, being sensitive to metabolic inhibition with SHAM/glycerol. Significant differences in drug transport were observed which could be correlated with resistance to isometamidium. The optimal pH for drug accumulation was lowered in the resistant trypanosomes; this finding, along with an observed change in specificity for the related compound homidium bromide, suggested that the specific receptor for isometamidium is altered in the resistant trypanosomes, possibly resulting in a reduction in drug uptake. In addition to these alterations in drug uptake, efflux of isometamidium also appears to occur in the resistant trypanosomes. Both a reduction in incubation temperature and metabolic inhibition increased the level of trypanosome-associated isometamidium in the resistant parasites. This was in contrast to observations using drug-sensitive parasites. Furthermore, the addition of calcium flux-modulating agents to the incubation medium also resulted in an increase in accumulation by the resistant parasites.

Following purification by affinity chromatography, three glutathione-binding proteins (TcGBP) of 45, 30, and 25 kDa were co-purified from Trypanosoma cruzi epimastigotes. Using 1-chloro-2,4 dinitrobenzene as substrate, a glutathione S-transferase activity of 70 nmol/min/mg of proteins was detected in the GSH binding fraction. An increased expression of TcGBP and total GST activity was observed upon incubation of parasites with phenobarbital, which is an inducer of GST synthesis. Immunofluorescence and electron microscopic experiments demonstrated that TcGBP were expressed by all developmental stages of the parasite, including infective forms. The expression of these proteins by intracellular dividing amastigotes could be in favour of a potential defensive role of these molecules against host attack. Results obtained by immunoprecipitation of in vitro translation products using anti-TcGBP antisera suggested that these three polypeptides are not glycosylated. In addition, antibodies directed against the TcGBP were found in a high proportion of T. cruzi-infected chronic chagasic patients' sera and in sera of chronically infected BALB/c mice. In contrast, acute chagasic patients' sera and acute-phase mouse sera were found to be poorly reactive with these proteins. Our results identify a new class of potential target antigens, which may be essential for the development of T. cruzi in its host. Their protective role in experimental models deserves to be investigated.

The morbidity and transmission dynamics of geohelminthiases are determined by the patterns of infection intensity in the community. Understanding the determinants of these patterns requires a combination of field, laboratory and theoretical study. Studies of age-specific reinfection, and of the phenomenon of predisposition, indicate that the major determinant of convex age-intensity profiles and of heterogeneity in infection intensity is the rate of establishment of infection, rather than the rate of adult worm mortality. The rate of establishment is, in turn, determined by exposure to, and protection from, infection. The evidence indicates that exposure, at least to the orally-transmitted geohelminths, varies with age and is highly heterogeneous between hosts. The immune response in geohelminthiasis is vigorous, parasite-specific, hetero geneous between hosts, and both age and infection dose dependent, but has yet to be convincingly shown to be protective. Since the immune response is itself a function of exposure, unravelling the interaction between ecology and immunology as determinants of geohelminth worm burden will require simultaneous assessment of both processes via immuno epidemiological study.

Dynamics of natural populations of Schistosoma mansoni were studied during 8 consecutive years among Rattus rattus populations from 8 transmission sites of the marshy forest focus of Guadeloupe (French West Indies). The schistosome population is over-dispersed (k = 0·119) within the murine hosts and ecological factors linked to the patchy environment may be responsible for such aggregated distribution. Analysis of the spatio-temporal variations in prevalences, intensities and abundances showed limited variations of the infection during the 8 years at the level of the whole parasite population but great spatial heterogeneity at the level of local schistosome populations. Inter-populational genetic variability linked to the degree of adaptation of this human parasite to the murine host may explain differences in transmission dynamics between the local populations of S. mansoni.

All instars of the pentastomid Porocephalus crotali, in the tissues of rat intermediate hosts and the lung of rattlesnake definitive hosts, are covered by a vesicular or stacked membranous secretory product which is synthesized in sub-parietal cells (SPC) and channelled to the cuticle via multitudinous chitin-lined ducts. In rats this enveloping foam of vesicles survives for the duration of a cuticle. We have purified and partially characterized the lipid and protein composition of lamellate droplets from SPC of infective nymphs. Lipids in the droplets comprised a mixture of neutral and polar lipid classes with cholesterol being the major neutral lipid and phosphatidylcholine the dominant polar lipid. In addition, Triton X-114 phase separation of the protein component of droplets partitioned these into an aqueous phase (with a major band at 60 kDa), a detergent phase with two hydrophobic polypeptides (24 and 16 kDa) and an insoluble pellet containing several minor proteins and major bands at 31 and 107 kDa. Western blotting, with rabbit anti-lamellate droplet antiserum, strongly label only the 60 kDa and the two hydrophobic proteins. Significantly no proteins at all label with serum from infected rats from 50–150 days post-infection, a finding endorsed by indirect fluorescent antibody tests on sectioned V–VII stage nymphs. Possible immunomodulatory functions of SPC-derived surface membranes are discussed and, in this regard, they are compared with pulmonary surfactant, a product of alveolar Type II cells which lines all vertebrate lungs.

The role of pulmonary cellular responses in the induction and expression of immunity to Schistosoma mansoni has been evaluated. From experiments in which mice were challenged at intervals after vaccination, we conclude that the resistance mechanism operating in the lungs develops between days 11 and 20. Injection of 51Cr-labelled splenocytes revealed that mononuclear cell recruitment to the lungs, stimulated by the arrival of attenuated schistosomula, intensified progressively between days 10 and 16 post-vaccination. The increased cellularity was reflected in a 19·5% augmentation in the wet-weight index (WW). The period of enhanced cell recruitment therefore coincided with the build-up of resistance. By day 22 post-vaccination infiltration had declined, whilst WW remained elevated. This indicates the persistence of recruited cells, which include schistosome-reactive T lymphocytes, in the lungs. We were unable to demonstrate augmented recruitment of 51Cr-labelled cells after challenge of vaccinated mice, but WW rose slightly, peaking on day 12. Although clearly of a lower order than the primary response, the secondary response was more rapid, implying the existence of immunological memory. These results accord with the concept that schistosome-reactive T lymphocytes recruited after vaccination ‘arm’ the lungs against the arrival of challenge parasites.

Having been trained at the London School of Hygiene and Tropical Medicine, I am a tropical medicine man in the tradition of Sir Patrick Manson, that is, a parasitologist. In his address inaugurating the London School of Tropical Medicine in 1899, entitled The Need for Special Training in Tropical Medicine, Manson stated that ‘the peculiar dis tribution of a large class of tropical diseases depends, in the first place, on the fact that they are entozoal diseases, in the second place, that the entozoa concerned require intermediate and definitive hosts, and, in the third, that one or other of these hosts requires a high atmospheric temperature, in other words are native to warm climates.’ He went on, ‘today the protozoan and the helminth, as regards tropical pathology, are in the ascendant.’ This belief still holds sway, as the great British and American tropical medical journals remain largely devoted to parasitic infections.

A contingency table approach was used to explore the influence of location, host species and time on the genetic composition of a Trypanosoma brucei population in Lambwe Valley, Kenya. Significant differences in zymodeme frequencies were noticed over comparatively short geographical distances suggesting that transmission of T. brucei is somewhat localized. A significant association was observed between zymodeme and the mammalian host from which T. brucei was derived. The association was consistent in different localities in Lambwe valley and remained stable for at least 32 months. These observations indicate that zymodemes are adapted to different host species and that genetic exchange has not disrupted host associations over this time-scale. A major change in the composition of the T. brucei population during a sleeping sickness outbreak in 1980 was confirmed. But while new zymodemes emerged, a decline in overall diversity was noted during times of high sleeping sickness incidence. The results can be explained by selection of T. brucei zymodemes for particular transmission cycles. Although it is not necessary to invoke genetic exchange, sex may help T. brucei to adapt to changes in selection pressures. Such a hypothesis helps to explain why T. brucei appears largely clonal in the short term, even though population studies indicate that sex is responsible for much genetic diversity in the long term. It also explains why neighbouring populations of T. brucei are composed of a different range of zymodemes formed from the same alleles. Such a view implies that genetic exchange has an important role in the microevolution of T. brucei populations.

Infection with digenetic trematodes causes an inhibition or complete cessation of fecundity in their intermediate hosts, freshwater snails. It has been demonstrated in the host–parasite combination Lymnaea stagnalis–Trichobilharzia ocellata that the action of the female gonadotropic hormones upon their target organs is inhibited by the peptide schistosomin. Schistosomin is produced in the central nervous system of the snail and released upon parasitic infection. In order to study the in vitro release of schistosomin, a bioassay was developed. Central nervous systems were incubated with either an acetic acid or a methanolic extract of larval stages of Trichobilharzia ocellata (miracidia, mother sporocysts, cercariae). The incubation media were chromatographed using HPLC and released schistosomin (-like material) was tested for bioactivity in the calfluxin bioassay. The in vitro release of schistosomin was only induced with a methanolic extract of cercariae. The nature of the cercarial factor is discussed.

To assess ectoparasite impact on individuals and populations of Gerbillus andersoni allenbyi under natural conditions, I addressed the following questions. Do ectoparasites affect their host fitness and, if so, how? Do ectoparasites affect host population level? Does this parasite–host interaction support the traditional concept of parasite evolution towards avirulence? For this purpose, host infestation, infection, survival, haematological indices, and physical condition were recorded. A field experiment which included manipulating host infestation while recording host survival was conducted to determine the causal relations between these variables. G. a. allenbyi was infested by 2 fleas (Synosternus cleopatrae and Stenoponia tripectinata), 5 mesostigmatid mites (Androlaelaps centrocarpus, A. hirsti, A. insculptus, A. marshalli and Hirstionyssus carticulatus), 1 tick (Rhipicephalus sanguineus), and 1 louse (Polyplax gerbilli). Ectoparasite burden significantly reduced host survival and red blood cell indices (red cell concentration, haemoglobin concentration and haematocrit). Ectoparasite burden did not significantly affect white blood cell concentration. Gerbils were not infected by haemoparasites or gut helminths which potentially could cause anaemia. The causal relationship between S. cleopatrae burden and host survival was established by manipulation of host infestation. Both ectoparasite removal and initial level of infestation significantly affected host survival. Ectoparasites that caused anaemia were not associated with host physical condition (PC), measured as weight/length3. None of the red blood cell indices was correlated with host PC. These results suggest both that host PC was not affected by ectoparasite burden and that exsanguination leading to anaemia was the main effect of the ectoparasites. Ectoparasite pressure on the host population (based on the ectoparasite effects as estimated by statistical models, combined with dispersion of the infestation within the host population) changed seasonally and was greatest when host density was the highest. A large segment of the gerbil population was affected by ectoparasites during the entire year. An explanation for the evolution of parasite virulence, contrasting parasites that evolve towards increased virulence with parasites that evolve towards avirulence is presented. This classification is primarily based on whether parasite impact is equated with a higher efficiency of host exploitation, or whether it is a ‘side effect’ of parasite biology.

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle.

Considerable diversity has been found in the reproductive behaviour of benedeniine (capsalid) monogenean parasites. Mating has been observed in Benedenia sp. 1 from the gills of Lutjanus carponatatus; externally attached spermatophores are not involved and parasites indulge in mutual cross-insemination with intromission. In contrast, there is evidence of spermatophore involvement in Benedenia sp. 2 from the fins of Lethrinus miniatus; mating was not observed but an individual was found carrying an external ovoid spermatophore attached by a stalk lodged in the vagina. In specimens of Benedeniella macrocolpa and B. posterocolpa, in which the male reproductive system was functional and the female system not fully developed, the everted cirrus was seen to be lodged in the parasite's own uterus, with, in some individuals, the tip of the cirrus inside the ootype. This is the first time that the copulatory organ has been observed inside the female reproductive tract of the same individual, not just in benedeniines but in monogeneans in general, and is also the first demonstration that monogeneans are capable of self-insemination via the uterine route.

Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z–Phe–Arg–MCA. Two of these were optimally active at alkaline pH, the other at pH 4·0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz–Arg–MCA readily, whilst the other cleaved Bz–Arg–MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukayrotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc–Ala–Ala–pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz–Arg–MCA hydrolase, the aminopeptidases and the Boc–Ala–Ala–pNA serine peptidase are cytosolic.

Methodology is described for the laboratory maintenance of the life-cycle of Sanguinicola inermis, using common carp (Cyprinus carpio) as the definitive host and Lymnaea peregra as the intermediate host. Lymnaea auricularia was also infected but is not considered to be a suitable laboratory host. Tench (Tinca tinca) were susceptible to infection at high cercarial doses but subsequent infection rates were low. Goldfish (Carassius auratus) and Lymnaea stagnalis were refractory to laboratory infection.

The pinworm (Oxyurida: Nematoda) guild in a laboratory colony of Periplaneta americana was investigated to answer the following questions. (1) Is guild structure stable with time? (2) Is there evidence of interaction among the species? (3) Is there evidence for niche diversification with respect to spatial distribution or developmental stage of host? Four species were found: Thelastoma periplaneticola, Thelastoma bulhoesi, Hammerschmidtiella diesingi and Leidynema appendiculatum; females of the first two could not be distinguished and they were lumped and referred to as Thelastoma sp.; 328 mid-instar hosts were dissected between September 1987 and February 1990, and the number of adult females of each pinworm species was recorded to investigate changes in guild structure through time. Seventy-five percent of hosts had Thelastoma sp., 62% had H. diesingi and 40% had L. appendiculatum; mean intensities were 6.5 (Thelastoma), 2.9 (H. diesingi) and 1.9 (L. appendiculatum). Relative numbers were stable with time and intensities of the species were similar whether they occurred alone in the host or in conjunction with other species. Thelastoma sp. and L. appendiculatum occurred together less often than expected and their numbers in individual hosts were negatively correlated. Both species occurred alone more often than expected. Surprisingly, H. diesingi and L. appendiculatum occurred together more often than expected. Intestines of 44 roaches were frozen in liquid nitrogen and divided into 5 equal sections to study longitudinal distributions of the species in the gut. Most L. appendiculatum and H. diesingi were found in the first, whereas most Thelastoma sp. occurred in the second segment. However, Thelastoma sp. dominated all segments of the gut. Worms were counted in 192 hosts belonging to 4 categories (early instar, late instar, adult male and adult female). Single species infections were more common in early instar hosts but there was no evidence that different species preferred different host stages. Modal distributions in male L. appendiculatum and T. bulhoesi suggest that these species may exhibit intraspecific interference competition similar to that observed in other pinworms. Such effects may be more important than interspecific effects in maintaining guild stability.

The ribosomal DNA (rDNA) unit of the liver fluke Opisthorchis viverrini has been cloned and characterized. The results demonstrated that the total length of this unit is approximately 13 kb, containing 4·2 kb of large subunit (LSU) rDNA, 2·0 kb of small subunit (SSU) rDNA, 1·0 kb of transcribed spacer DNA and 5·8 kb of non-transcribed + external transcribed spacer DNA. Examination of the non-transcribed spacer region between different rDNA units showed variation in the restriction sites rather than in the length. Judging from hybridization of the rDNA plasmid to the restriction endonuclease digest of genomic DNA, rDNA units represent 6·1% of the total genomic DNA. At the RNA level, the LSU rRNA of O. viverrini and Fasciola gigantica contained a hidden break. The molecule consisted of two fragments co-migrating with a SSU rRNA, when electrophoresis was carried out under denaturing conditions. Ribosomal RNA sequence comparison has been previously used to determine phylogenetic classification of parasitic organisms. The sequence of 381 nucleotides at the 5′ terminus of the LSU rRNA gene was determined and compared with those from species previously reported by other investigators. Phylogenetic classification of O. viverrini, as determined by rRNA gene sequence comparison, is comparable with the conventional taxonomic classification.

Ultrastructural investigations (SEM, TEM) combined with lectin-binding analysis, have revealed concurrent modifications in tegumentary structure and surface glycoconjugates during the establishment and differentiation of Echinococcus multilocularis metacestodes in jirds. The laminated layer, which is amorphous and rich in polysaccharides when initially secreted by the young cyst, takes on a different appearance and has a different glycoconjugate composition according to whether the cyst becomes fertile or sterile. The laminated layer of fertile cysts transforms into a microfibrillar matrix, the protein content of which may increase while sugar content decreases during protoscolex differentiation. Independently of this structure, brood capsules, from which arise protoscoleces, are formed by invagination of the cyst tegument. The intense secretion of glycoconjugates from the brood capsule wall during invagination may serve to interact with host factors passing through the laminated layer. The combined use of ultrastructural study and lectin labelling has allowed the demonstration of an ultrastructural and biochemical gradient of differentiation of the protoscolex. Seven stages of differentiation have been described. The possibility that the excreted–secreted tegumentary glycoconjugates, revealed by lectin labelling during protoscolex differentiation, might be the gradual biochemical expression of one or several stimuli implicated in the phenomenon of protoscolex maturation, is discussed.

Entomopathogenic nematodes of the genus Heterorhabditis are morphologically conservative, consequently the majority of isolates remain unassigned at the species level. When a Heterorhabditis infective juvenile infects an insect host, it develops into a protandrous hermaphrodite female. These first-generation females give rise to a second generation which contains both males and females. Because of this complex life-cycle and also because of uncertainty as to whether second-generation females are amphimictic, cross-breeding studies to facilitate species determination have not been carried out previously. We demonstrate here that second-generation Heterorhabditis females are amphimictic. Because of this finding, we have been able to develop a successful cross-breeding technique for the purposes of species determination in Heterorhabditis. Interstrain crosses using second-generation males and females from the appropriate strains have been successfully set up in Xenorhabdus luminescens-treated G. mellonella cadavers and also on agar plates. Using the techniques described here we confirm that H. bacteriophora (Brecon strain), H. megidis and H. zealandica are distinct biological species, we note that the H. bacteriophora group contains at least 2 species and we provide evidence for the existence of a new Irish species of Heterorhabditis.