Continuous culture was achieved in several cell lines of a microsporidium obtained from the skeletal muscle of an AIDS patient. Development in COS-1 and RK13 cells was prolific. Spores from the original biopsy were also inoculated into athymic mice by i.m. and i.p. routes. Infection was found in several organs as well as in skeletal muscle after a few weeks. All stages were surrounded by an electron-dense surface coat. Meronts had 2–4 nuclei and divided by binary fission. In sporogony the surface coat became separated from the plasma membrane to form a sporophorous vesicle, within which division into sporoblasts was effected by repeated binary fissions. The number of sporoblasts (and later spores) within the sporophorous vesicles varied from 2 to > 32 and the sizes of the vesicles varied, according to the number of spores contained therein, from 5 μm diameter to 14·0 × 11·0 μm. Spores measured 4·0 × 2·4 μm and had a prominent posterior vacuole. The parasite differs from the genus Pleistophora in that it does not form multinucleate sporogonial plasmodia and that the sporophorous vesicle enlarges during sporogony and its wall is not a multilayered structure. It is proposed to place it in a new genus and species Trachipleistophora hominis n.g., n.sp.

The migration of the schistosomula of Schistosoma mansoni labelled with [75Se] has been followed from the skin to the hepatic portal system. Parasites were detected in all mouse tissues by compressed organ autoradiography. Two separate experiments were performed to track parasites in normal mice, and in mice previously vaccinated with irradiated cercariae. In normal mice, the profile of numbers of autoradiographic foci detected in the skin, lungs, systemic and splanchnic organs was described with time post-infection. The distribution of parasites to systemic organs, following exit from the lungs, paralleled the fractional distribution of cardiac output. Accumulation of schistosomula in the hepatic portal system was complete by day 21 post-infection. Only 2–3 passes of parasites around the vascular system would be required to produce the hepatic portal population. No significant decline in total foci was detected in the first 12 days post-infection. The majority of parasite elimination appeared to occur in the lungs as late as day 21, with lesser proportions in the systemic organs and skin infection site. The pattern of migration in vaccinated mice was similar to that in normal animals. One difference observed was the longer duration of stay in the skin; however, the majority of parasites eventually reached the lungs. The systemic phase of migration occurred on a reduced scale, as did accumulation of parasites in the hepatic portal system. The decline in total foci in vaccinated mice commenced approximately 7 days earlier than in normal mice and proceeded to a lower end-point. Again the majority of parasite elimination appeared to occur in the lungs with lesser proportions in the systemic organs and skin infection site. It is suggested that resistance to reinfection in vaccinated mice has two additive components which combine to retard the migration of schistosomula within the vasculature, preventing them from reaching the hepatic portal system.

This paper examines the efficiency of acquired resistance in protecting the fish host, rainbow trout (Oncorhynchus mykiss), against the trematode parasite Diplostomum spathaceum, and the hypothesis that fish recognize areas where infective stages are aggregated and show avoidance behaviour. We found that when fish with a low level of infection were held in restricted cages in natural conditions they became infected and developed cataracts as a result of this infection. This suggests that acquired resistance is insufficient in protecting fish against the parasite or the deleterious effects of infection in conditions where fish could not avoid the parasite. Behavioural experiments in the laboratory showed that fish reacted to the parasite cercariae by avoiding the infection source, which decreased the rate of parasite establishment. We conclude that by using a combination of behavioural avoidance and physiological resistance, fish could defend against the parasite more effectively.

This review addresses the role of compounds secreted into the external environment that mediate important aspects of tick behaviour. Known as semiochemicals, these information-containing compounds include pheromones (used for conspecific communication), allomones (defence secretions) and kairomones (used for host identification and location). An impressive body of knowledge has accumulated concerning the identification of the compounds that comprise these semiochemicals. Pheromones are the best known and intensively studied, including arrestment (=assembly) pheromones, attraction–aggregation–attachment (AAA) pheromones and sex pheromones. Arrestment behaviour is mediated by contact with excreta from other ticks. In contrast, aggregating and sexual behaviours comprise a hierarchy of responses to different types of chemical compounds that must be recognized in a sequential order to achieve the end result. Ixodid ticks also produce an allomone that protects against certain insect predators. Finally, ticks use kairomones for host identification, e.g. volatiles such as CO2 and NH3 and various oils such as glandular secretions from deer. Knowledge of tick pheromones has been used to develop innovative new technologies for tick control. These products incorporate tick pheromones and small amounts of pesticide to attract and kill ticks on their hosts or in vegetation. The kairomones and the tick allomone also may be of interest for use in controlling ticks.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3α, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6·6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co- infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5–14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Previous studies in deliberately infected sheep have shown an association between IgA activity against 4th-stage larvae of Teladorsagia circumcincta and parasite growth, development and fecundity. The purpose of this research was to determine if these results could be confirmed in naturally infected sheep and to explore the hypothesis that plasma IgA activity could help to identify resistant lambs with shorter adult nematodes. Plasma IgA activity was skewed with most animals having relatively low levels of IgA activity. Plasma IgA activity was repeatable and highly heritable. Animals with increased IgA activity had lower egg counts and shorter adult female T. circumcincta. Therefore, under conditions of natural parasite challenge, plasma IgA activity may help to identify lambs resistant to T. circumcincta.

Efficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

The oncomiracidium of the monogenean skin parasite Entobdella soleae finds its flatfish host, Solea solea, by chemoreception. The free-swimming larva responds to a specific substance secreted by the skin of the common sole, attaches itself to this skin and immediately sheds its ciliated epidermal cells. Larvae respond in the same way to agar jelly which has been in contact with the skin of S. solea.

The oncomiracidia attach to S. solea skin in preference to that of other soleid fishes (Buglossidium luteum and Solea variegata), pleuronectid fishes (Limanda limanda, Pleuronectes platessa) and elasmobranch flatfishes (Raia spp.).

Larvae respond strongly to isolated epidermis from Solea solea but show no response to the fish's cornea, indicating that the attractive substance is produced by the mucus cells in the fish's epidermis.

The larvae attach with equal readiness to skin from the upper and lower surfaces of S. solea. Thus the preponderance of young parasites on the upper surfaces of soles is due not to a preference for the upper skin but to the fact that the lower skin is in contact with the substratum and cannot be reached by the larvae.

These results led to speculations on the way in which host specificity evolved in the Monogenea.

I am indebted to Mr J. E. Green of the Plymouth Laboratory for setting up a tank containing infected soles and for maintaining the tank and feeding the fishes for many months.

I am also grateful to the Directors and Staff of the Plymouth Laboratory and the Fisheries Laboratory, Lowestoft, for hospitality and assistance. I am particularly grateful to Mr J. Riley of the Lowestoft Laboratory for providing various flatfishes.

This review examines the phenomenon of co-feeding transmission in tick-borne pathogens. This mode of transmission is critical for the epidemiology of several tick-borne viruses but its importance for Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, is still controversial. The molecular mechanisms and ecological factors that facilitate co-feeding transmission are therefore examined with particular emphasis on Borrelia pathogens. Comparison of climate, tick ecology and experimental infection work suggests that co-feeding transmission is more important in European than North American systems of Lyme borreliosis, which potentially explains why this topic has gained more traction in the former continent than the latter. While new theory shows that co-feeding transmission makes a modest contribution to Borrelia fitness, recent experimental work has revealed new ecological contexts where natural selection might favour co-feeding transmission. In particular, co-feeding transmission might confer a fitness advantage in the Darwinian competition among strains in mixed infections. Future studies should investigate the ecological conditions that favour the evolution of this fascinating mode of transmission in tick-borne pathogens.

The last 30 years have seen an impressive use of ultrasonography (US) in many fields of veterinary and clinical medicine and the technique is being increasingly applied to a wide variety of parasitic infections including the cestode zoonoses Echinococcus granulosus and E. multilocularis. US provides real-time results which are permanently recordable with a high resolution and diagnostic accuracy. These properties, coupled with the clinical value of the images obtained and the non-invasive nature of the test which is safe, require no special patient preparation time; it is easy to operate and this has resulted in the establishment of US as the diagnostic technique of choice for cystic (CE) and alveolar (AE) echinococcosis. The lack of ionizing radiation and side-effects mean that examination times are not restricted. The hand-held probes facilitate what amounts to a rapid, bloodless non-invasive laparotomy, enabling a search from an infinite number of angles for lesions producing information on their number, size and type of cysts, their location and clinical implications. Such clinical information has facilitated the development of treatment protocols for different cyst types. Less invasive surgical techniques, such as US guidance for PAIR (Puncture, Aspiration, Injection, Re-aspiration), PAIRD (PAIR plus Drainage) or PPDC (Percutaneous Puncture with Drainage and Curettage) are also possible. Longitudinal US studies have facilitated monitoring the effects of the outcome of treatment and chemotherapy. Portable ultrasound scanners which today weigh as little as a few pounds, powered by battery or generators have facilitated the use of the technique in mass community-based screening studies. The majority of these studies have been conducted in remote, low socio-economic areas where there were few, if any, hospitals, veterinary facilities, schools or trained personnel. The surveys led to the discovery of unexpectedly high prevalences of CE and AE in asymptomatic individuals of endemic areas and especially amongst transhumant or nomadic pastoralists living in various parts of the world. Screening for CE and AE is justified as an early diagnosis leads to a better prognosis following treatment. The application of US in field and clinical settings has led to a better understanding of the natural history of CE and AE and to the development of a WHO standardized classification of cyst types for CE. This classification can be used in helping define the treatment options for the different cysts found during the surveys, which in turn can also be used to calculate the public health cost of treating the disease in an endemic community. The case mix revealed can also influence the specificity (particularly proportions of cyst types CE4 and CE5 and cystic lesions – CL) of US as a diagnostic test in a particular setting. Community based US surveys have provided new insights into the public health importance of CE and AE in different endemic settings. By screening whole populations they disclose the true extent of the disease and reveal particular age and sex risk factors. Through the treatment and follow-up of all infected cases found during the mass screening surveys a drastic reduction in the public health impact of the disease in endemic communities can be achieved. Educational impacts of such surveys at the national, community and individual levels for both professional and lay people are beginning to be appreciated. The translation of the information gained into active control programmes remains to be realized. In areas where intermediate hosts, such as sheep and goats, are not slaughtered in large numbers mass US screening surveys to determine the prevalence of CE in livestock has proved possible. Longitudinal studies in such intermediate hosts would reveal changes in prevalence over time, which has been used as a marker for control success in other programmes. Mass US screening surveys in an ongoing control programme in Argentina has demonstrated the early impact of control in the human population and identified breakthroughs in that control programme. Mass US screening surveys must adhere to the highest ethical standards and the outcome of surveys should result in the application of appropriate WHO recommended treatment options for different cyst types. Follow-up strategies have to be in place prior to the implementation of such surveys for all infected individuals who do not require treatment and for all suspected, but not confirmed, cases found during the surveys. The use of US in community screening surveys has revealed the complexity of ethical issues (informed consent, confidentiality, follow-up, detection of lesions that are not the focus of the study etc) and also provided real solutions to providing the most ethical guidelines for the early detection and treatment of CE and AE.

A simple, quick and reproducible method consisting of density-gradient centrifugation of homogenized infected mouse brain tissue on Percoll is described for the isolation and purification of cysts of Isospora (Toxoplasma) gondii. A 100% recovery of cysts, with 74·2% in a single fraction with a specific gravity of 1·056, was obtained by overlaying homogenates of infected mouse brains on a pre-formed Percoll gradient and centrifugation at low g forces. With this procedure recovery was independent of the age of the cysts. Titration of purified cystozoites showed there to be no loss of infectivity.

Fitness of tick-borne pathogens may be determined by the degree to which their infection dynamics in vertebrate hosts permits transmission cycles if infective and uninfected tick stages are active at different times of the year. To investigate this hypothesis we developed a simulation model that integrates the transmission pattern imposed by seasonally asynchronous nymphal and larval Ixodes scapularis ticks in northeastern North America, with a model of infection in white-footed mice (Peromyscus leucopus) reservoir hosts, using the bacteria Borrelia burgdorferi and Anaplasma phagocytophilum as examples. In simulations, survival of microparasites, their sensitivity to reduced rodent and tick abundance, and to ‘dilution’ by a reservoir-incompetent host depended on traits that allowed (i) highly efficient transmission from acutely-infected hosts, (ii) long-lived acute or ‘carrier’ host infections, and/or (iii) transmission amongst co-feeding ticks. Minimum values for transmission efficiency to ticks, and duration of host infectivity, necessary for microparasite persistence, were always higher when nymphal and larval ticks were seasonally asynchronous than when these instars were synchronous. Thus, traits influencing duration of host infectivity, transmission efficiency to ticks and co-feeding transmission are likely to be dominant determinants of fitness in I. scapularis-borne microparasites in northeastern North America due to abiotic forcings influencing I. scapularis seasonality.

According to the World Health Organization the global estimate of malaria is over 200 million infections, the majority of which are caused by the most life-threatening species, Plasmodium falciparum (Report of the Steering Committees of the Scientific Working Groups on Malaria, World Health Organization, June 1983). The causative agent of the disease, the malarial parasite, requires two hosts: a blood-sucking mosquito and a blood-containing vertebrate. Commonly, infection of the vertebrate begins when an infected mosquito bites a suitable vertebrate and injects minute sporozoites into the bloodstream. Within 30 mm the introduced sporozoites leave the bloodstream and enter parenchymal cells of the liver (mammals) or endothelial cells (birds). In these sites the parasite undergoes asexual multiplication (= exo-erythrocytic schizogony) producing daughter progeny called merozoites. The exo-erythrocytic merozoites are released from the tissues into the circulation where they invade red blood cells. Within an erythrocyte the merozoite undergoes asexual multiplication (= erythrocytic schizogony) producing a substantial number of merozoites. The erythrocyte lyses, merozoites are released, and invasion of another erythrocyte may then take place. The synchronous rupture of the red cell and merozoite release is marked by the periodic fever–chill cycles so characteristic of the malarial infection. Some merozoites continue to reinvade other erythrocytes and multiply by asexual means, whereas others enter erythrocytes and differentiate into sexual stages, male or female gametocytes. When a suitable mosquito feeds on an infected vertebrate gametocytes are ingested and the sexual cycle of development is initiated. In the mosquito stomach the gametocytes transform into gametes, fertilization takes place, the resultant worm-like zygote penetrates the cells of the mosquito gut and comes to lie on the outer surface of the stomach. Here each zygote forms a cyst-like body, the oocyst, within which thousands of sporozoites are produced by asexual multiplication. When the swollen oocysts burst, sporozoites are freed and these make their way to the salivary gland. At the next blood feeding the mosquito injects the infective sporozoites and the life-cycle is completed.

The nuclear DNAs of 71 trypanosome stocks from different African countries, representative of the three Trypanosoma brucei subspecies, and one T. evansi stock, have been analysed by the combined use of restriction endonuclease digestion, gel electrophoresis and molecular hybridization with both trypanosome surface-antigen-specific and undefined genomic DNA probes. In contrast with T. brucei brucei and T. brucei rhodesiense stocks, all the T. b. gambiense stocks are characterized by a conserved, specific DNA band pattern, regardless of the probe. This allows T. b. gambiense to be non-ambiguously identified. On the contrary, T. b. brucei and T. b. rhodesiense, which could not be discriminated by the same criteria, both yield highly variable DNA band patterns. Our data confirm that domestic animals like pig, dog and sheep constitute a potential reservoir for T. b. gambiense. Using a numerical analysis of the DNA hybridization patterns we have measured the degree of similarity between the 72 trypanosome stocks. This investigation shows that all T. b. gambiense stocks are included in the same homogeneous population, while the stocks from the two other subspecies seem to be distributed in several heterogeneous groups, some of these showing correlation with the geographical origin of the trypanosomes. It is concluded that (i) T. b. gambiense stands out as a real subspecies that has undergone a distinct evolution relative to the ‘non-gambiense’ group, (ii) the alleged T. b. rhodesiense subspecies does not fit with any of the groups evidenced by our cladistic analysis and hence does not appear as a distinct subspecies and (iii) ‘non-gambiense’ trypanosomes are probably evolving much more rapidly than T. b. gambiense. Different aspects of trypanosome relationships and evolution are discussed.

Three techniques for estimating the intensity of Opisthorchis viverrini infection in individuals from a Northeast That community are compared. Egg counts were determined using a quantitative formalin/ethyl acetate technique, worm burdens were estimated by expulsion chemotherapy and antibody levels were measured by ELISA. Log-transformed worm and egg counts were closely correlated (r = 0·80), suggesting that both measurements provide good assessments of relative intensity of infection. However, no Opisthorchis worms were recovered from 34 people with high egg counts; probably due to problems with the expulsion technique in some individuals. Examination of egg production per fluke indicated that each fluke contributed an average of 180 eggs per gram (epg) of faeces and fecundity was negatively associated with total worm burden. Serum IgG levels correlated significantly with Opisthorchis egg count (r = 0·61) at two independent assessments. Although significant associations were observed between antibody levels and echinostome infection, analysis suggested that these reflected independent associations between these two variables and Opisthorchis infection and age. We conclude that all three measurements are useful for epidemiological studies.

There is some evidence that pathology induced by heavy malaria infections (many oocysts) increases mortality of infected mosquitoes. However, there is little or no published evidence that documented changes in feeding behaviour associated with malaria infection also contribute to higher mortality of infected mosquitoes relative to uninfected individuals. We show here for the first time that, in a natural situation, infection by the sporozoites of the malaria parasite Plasmodium falciparum significantly reduced survival of blood-feeding Anopheles gambiae, the major vector of malaria in sub-Saharan Africa. To estimate feeding-associated mortality of infected mosquitoes, we compared the percentage of sporozoite infection in host-seeking mosquitoes caught before and after feeding. The infection rate was 12% for mosquitoes caught during the night as they were entering a tent to feed; however, only 7·5% of the surviving members of the same cohort caught after they had had the opportunity to feed were infected. Thus, Plasmodium falciparum sporozoites increased the probability of dying during the night-time feeding period by 37·5%. The increase in mortality was probably due to decreased efficiency in obtaining blood and by increased feeding activity of the sporozoite-infected mosquitoes that elicited a greater degree of defensive behaviour of hosts under attack.

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.

Both histological and histochemical studies have been made of the food, mode of feeding, gut cell structure and digestive sequences in a selected number of digenetic trematodes.

The species investigated range from gut-dwelling trematodes feeding on the superficial epithelial tissues and associated mucoid secretions of the host to those forms living within the respiratory and circulatory system and feeding exclusively on blood.

The mode of feeding is suctorial, brought about by the muscular pharynx and normal attachment process of the oral sucker. In one case there is evidence that this purely mechanical process is supplemented with enzymic secretions produced by the trematode which have a histolytic effect upon host tissues.

On the basis of gut cell structure the species investigated are divided into those with cells and associated microvilli that vary in size and appearance so that the gastrodermal border is irregular in outline, and those with a gastrodermis comprising regular cuboidal or columnar cells bearing microvilli organized into a striated border. There is evidence to suggest that this difference in gut cell structure reflects differences in digestion. In all cases, gland cells are absent from the gastrodermis which is capable of both secretory and absorptive functions.

Digestion is predominantly an extracellular process, but the exact sequence varies according to the nature of the food ingested and different degrees of adaptation are shown by the trematodes to the blood-feeding habit. The tissue-feeding species show less modification.

I wish to thank Dr J. B. Jennings for helpful discussion and advice throughout the course of this work. The study was undertaken during the tenure of a Studentship from the Department of Scientific and Industrial Research.

Circumstantial evidence suggests that the earlier detachment of Boophilus microplus larvae from highly resistant cattle follows the release of histamine at the attachment site. In vivo and in vitro experiments show that a proportion of the larvae will detach following injection or infusion of histamine. Other mediators such as bradykinin, prostaglandin E2, 5-hydroxytryptamine and dopamine have little or no effect on tick behaviour in vivo. Sensitivity to histamine declines as larval attachments stabilize, and repeated injections have no effect on the weight of larvae after 3 days on the host. Response to histamine is discussed in relation to host resistance, histology of the feeding lesion and larval behaviour.