An in vitro culture of Plasmodium falciparum (isolate FCUP-1/RSA) was arrested in the trophozoite stage with α-difluoromethylornithine (DFMO), followed by sorbitol treatment to liberate intracellular parasites from infected erythrocytes. Most of the unlysed erythrocytes and ghost membranes were removed by filtration, after agglutination with anti-erythrocyte monoclonal antibodies or wheat-germ agglutinin. A highly purified parasite preparation was obtained after chromatography on an immuno-affinity column consisting of polystyrene particles to which anti-erythrocyte monoclonal antibodies had been adsorbed. Isolated parasites were free of surrounding erythrocyte membranes and structurally intact as assessed by transmission electron microscopy, SDS–PAGE and ELISA.

The frequency of recombination between unlike genotypes is central to understanding the generation of genetic diversity in natural populations of malaria. Here we suggest a way of investigating the problem which could complement conventional biochemical approaches to the population genetics of malaria. Sex allocation theory is one of the most successful areas of evolutionary biology. A well-supported prediction is that progressively less female-biased sex ratios are favoured with more outcrossing; equal numbers of males and females being evolutionarily stable in randomly mating outbred populations. We present a simple game theory model to support the idea that outcrossing rates in malaria will be correlated with the sex ratio of gametocytes in the peripheral blood of vertebrate hosts. Blood films from epidemiological surveys and culture-adapted isolates from Madang Province, Papua New Guinea, were used to estimate average gametocyte sex ratio of Plasmodium falciparum in the area. The geometric mean proportion of males in the population was 0.18 (95% confidence limits: 0.15–0.22). From our model, we estimate that, on average, 36% of zygotes are the result of outcrossing. This estimate assumes that most microgametes released following exflagellation are capable of fertilization. If, on average, fewer than about 70% of microgametes are capable of fertilization (as is the case in at least one other species of Plasmodium), the observed sex ratio would be consistent with between zero and 36% of zygotes being the result of outcrossing. These estimates suggest that there is usually a numerically dominant genotype in the gametocyte population in a blood meal, and that a considerable amount of selfing is occurring in P. falciparum populations in the Madang region, even though it is an area of intense year-round transmission.

A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650–800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range. The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock. G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA. Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4. The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite. In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture. Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species of Eimeria which infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility to E. tenella and susceptibility to the other species: lines which produced most oocysts of E. tenella produced least oocysts of the other species and vice-versa.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I – most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from ‘super-susceptible’ cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf–). In these cats the peak mf levels were seldom above 10000 mf/ml. Group II – these cats had less than 10 000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III – these cats became mf – during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics. Group IV – in these cats there was a sudden decline in the number of mf, usually in the first year and after they have received a relatively small number of re-infections. Circulating adult B. pahangi antigen disappeared from the serum within a short time of the loss of mf and at autopsy no adult female worms were found. Group V – these cats do not become mf+ when infected. These groups are compared to the parasitological groups seen in areas endemic for infection with Wuchereria bancrofti.

The cDNA probe, Lmet2, was labelled with digoxigenin and used in a chemiluminescent system to detect fewer than 100 membrane-immobilized Leishmania parasites. The probe was found to hybridize primarily with members of the L. donovani complex but a slight cross-reaction was also observed with greater than 5 x 104L. major. This cross-reaction was reduced by hybridizations in 50% formamide at 37 °C. Formamide also significantly reduced non-specific binding of the digoxigenin-labelled probe to the membrane support which, in hybridizations without formamide, masked the specific hybridization signal. This background was not observed with the corresponding radio-isotope labelled probe. With hybridizations in formamide the sensitivity achieved by the chemiluminescent system after exposure to film for 3 h was greater than that achieved by the isotopic system even after autoradiography for 24 h.

Naturally occurring immunoglobulin M antibodies to Toxoplasma gondii in human sera were examined for their ability to enhance toxoplasmacidal activities of human neutrophils to show a role of natural antibodies in immunity to the early stage of Toxoplasma infection in humans. Neutrophils were mixed with tachyzoites at a ratio of 1:5 in the presence or absence of antibodies and were cultured for 1–18 h for microscopical examination. The count of tachyzoites phagocytosed in 200 neutrophils within 1 h was significantly higher in the presence of sera with natural IgM antibody levels of > 0.8 than those of < 0.1, with 2.4- to 2.9-fold differences (P < 0.02). The total tachyzoite counts at 18 h decreased to 26–39% of those at 1 h in the presence of natural IgM antibody levels of > 0.8 (P < 0.01), while the counts at 1 h consistently increased by 18 h in the absence of natural IgM antibodies. These results indicate that relatively high levels of natural IgM antibodies enhanced phagocytic and microbicidal activities of neutrophils against Toxoplasma. The enhancement was dose dependent, and was also weaker than that obtained with the same dilution of sera from individuals with chronic or acute infections. Live and dead tachyzoite counts in infected neutrophils suggested a quicker effect of natural IgM antibodies than IgG antibodies elicited by infection.

Patients infected with Schistosoma mansoni showed an abnormal response to a test dose of tryptophan, with little increase in the urinary excretion of kynurenine, hydroxykynurenine, xanthurenic and kynurenic acids, N1-methyl nicotinamide, methyl pyridone carboxamide, 5-hydroxytryptamine or 5-hydroxyindoleacetic acid. In contrast to previous reports, this is different from the pattern of tryptophan metabolism seen in vitamin B6 deficiency. Furthermore, the patients' plasma concentrations of pyridoxal phosphate were within the reference range, and supplementation for 5 days with 20 mg vitamin B6/day did not affect tryptophan metabolism. Treatment with a single dose of Praziquantel resulted in a substantial restoration of normal tryptophan metabolism. In mice infected with S. mansoni there was a similar impairment of tryptophan metabolism, as shown by considerably reduced formation of 14CO2 after the administration of a tracer dose of [14C]tryptophan. Again, the administration of vitamin B6 supplements did not correct tryptophan metabolism in the mice. Treatment with Praziquantel resulted in substantial restoration of the production of 14CO2 from [14C]tryptophan. There was no evidence of vitamin B6 deficiency (as determined by erythrocyte aspartate aminotransferase activation coefficient) associated with infection in the mice, although there was a redistribution of pyridoxal phosphate between tissues, with a reduction in the concentration of liver, spleen and kidney, and an increase in skeletal muscle.

The prevalence of Schistosoma mansoni infections of Biomphalaria pfeifferi shows seasonal variation. Field data from Zimbabwe show annual ranges from 0 to 7%. In this paper a mathematical model of B. pfeifferi population dynamics and S. mansoni epidemiology is used as a framework for analysis of these patterns. Snail fecundity is a function of snail age and of temperature, and is apparently affected by other seasonal factors. The pre-patent period is dependent on temperature. Infection affects snail fecundity and mortality. Parameter values are derived from previous field and laboratory studies. The force-of-infection is estimated from the analysis of size-prevalence data. Using observed temperatures, model output agrees well with field data on snail abundance and prevalence of infection over a 14-month period. Seasonal variation in prevalence largely reflects variation in the pre-patent period and in snail population age structure. The possible role of seasonality in the force-of-infection is discussed. Prevalence patterns are not greatly affected by year-to-year differences in temperature. Significant seasonal variation in snail–man transmission rates is expected.

Faecal egg counts are often used to measure Schistosoma mansoni infection, but the considerable variation between successive counts complicates their interpretation. The stochastic model described in this paper gives a description of observed egg counts in a population and can be used as a tool to gain an insight into the underlying worm load distribution. The model distinguishes between two sources of variation in egg counts: (1) variation caused by the difference in worm load between individuals, and (2) the variability of egg counts for an individual with a given worm load. Empirical data, single and repeated measurements, from surveys in five villages in Burundi and Zaire have been used to fit and validate the model. We have discussed possible mechanisms that explain the differences in estimated values between the villages. The model indicates that the expected number of eggs in a stool sample per S. mansoni worm pair is lower than suggested by autopsy data and that, possibly as a consequence of immunity, the inter-individual variation in worm loads decreases with age.

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.

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.

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.

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.

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.

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.

The importance of host heterogeneity in generating aggregation was investigated using Heligmosomoides polygyrus (Nematoda) in laboratory mice. Parameters of infection were compared between inbred and outbred mice, between primary and challenge infection protocols, and between gavage and natural exposure protocols, to investigate the relative effects of innate resistance, acquired resistance and behaviour, respectively. Heterogeneity in acquired resistance was identified as the most consistent factor leading to variability and aggregation of H. polygyrus numbers in mice. This hypothesis was supported in two experiments where groups of mice did not develop resistance to challenge infection (use of certain inbred strains of mice and immunosuppression with corticosteroids in the drinking water) and where variability in worm numbers after the challenge infection was comparable with that after the primary infection. Heterogeneity in host behaviour, particularly in behaviours enhancing skin contact with larvae, also was associated with increased heterogeneity in worm burden, though not as consistently as heterogeneity in acquired resistance. Surprisingly, worm burdens were not more variable in outbred compared with inbred mice. Our data suggest that the relative contributions of innate resistance, acquired resistance and behaviour in generating variable worm burdens are likely to vary spatially and temporally.

Cyclosporin A (CsA) induced significant changes in parasite morphology when administered to mice infected with Hymenolepis microstoma. Gross morphological damage consisted of proglottis swelling and the formation of protuberances from the worm surface, visible with a low-power dissecting microscope, occurring most frequently in the posterior third of the strobila. Gross morphology and ultrastructure were examined further using scanning and transmission electron microscopy. Swollen proglottides exhibited areas covered in small pits and fissures (diameter approximately 1–2 μm) but it was not possible to establish the significance of this damage. The brush-border and distal cytoplasm appeared largely intact although some evidence of swelling of the basal membrane invaginations and possible fluid accumulation was seen in drug-treated TEM sections. The apparent oedematous condition of many of the proglottides from drug-treated mice may indicate that CsA treatment mediates permeability changes in the worm surface membrane but the mechanisms by which this may occur remain to be elucidated. The effects of CsA on the morphology of H. microstoma correlate with the previously described anthelmintic activity of the drug against this parasite where CsA treatment dramatically reduces worm growth, retards migration into the bile duct and limits parasite survival.

A double-blind placebo trial was conducted to determine the effect of moderate to high loads of Trichuris trichiura (whipworm) infection on the cognitive functions of 159 school children (age 9–12 years) in Jamaica. Infected children were randomly assigned to Treatment or Placebo groups. A third group of randomly selected uninfected children were assigned to a Control for comparative purposes. The improvement in cognitive function was evaluated using a stepwise multiple linear regression, designed to control for any confounding variables. The expulsion of worms led to a significant improvement in tests of auditory short-term memory (P < 0.02; P < 0.01), and a highly significant improvement in the scanning and retrieval of long-term memory (P < 0.001). After 9 weeks, treated children were no longer significantly different from an uninfected Control group in these three tests of cognitive function. The removal of T. trichiura was more important than Ascaris lumbricoides in determining this improvement. The results suggest that whipworm infection has an adverse effect on certain cognitive functions which is reversible by therapy.

The distribution of the fluorescent ivermectin derivative (4″ 5,7 dimethyl bodipy proprionylivermectin, referred to here as bodipy ivermectin) was studied in muscle vesicle membranes from the parasite Ascaris suum. Incubation in bodipy ivermectin, at concentrations greater than 1 ng/ml, produced a dose-dependent fluorescence in the vesicles. The level of fluorescence was not depressed (but increased) by prior incubation with 1 μM unlabelled ivermectin. This observation suggested that most of the bodipy ivermectin was free in the lipid phase of the membrane and not associated with a receptor. The trypan blue quenching technique showed that the bodipy ivermectin did not cross the membrane bilayer but remained in the outer monolayer. Lateral mobility of the probe in the membrane was studied using the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficient (DL) was 14.0 x 10−10 cm2/s which was nearly three times faster than the lipid probe octadecanoyl-aminofluorescein in membranes from the same preparations; only 46% of the probe was mobile, showing that within a 1.2 μm radius the distribution of the bodipy ivermectin in the membrane was not homogeneous. These observations are used to suggest that: the site of action of livermectin is located in the outer monolayer of the membrane (possibly on the Cl− channel); and that the speed of onset of action in isolated membrane preparations may be limited by diffusion in the membrane.