The rediscovery of Elleipsisoma thomsoni Franca 1912 from Talpa europaea is reported. This organism, whose identity as a parasite was initially doubted, is shown to be an intra-erythrocytic protozoan parasite. Light microscope observation confirmed the original description and 22/53 (42·5%) moles examined from a locality near Blackpool were positive. Organ impression smears demonstrated that E. thomsoni was concentrated in the heart and lungs, occasionally in the kidneys, liver and spleen. Electron microscope studies conclusively showed that the parasite was a member of the phylum Apicomplexa. A typical apical complex was present with rhoptries, micronemes, polar ring and microtubules but, in contrast to the genera Plasmodium, Babesia and Theileria, a conoid was also present. The parasitized red cells were larger than normal mature erythrocytes and the remaining cytoplasm of the erythrocyte was less dense than that of non-infected red cells. The problem of studying this enigmatic parasite and its life-cycle are described.

Following recent suggestions that a peaked host age–parasite abundance curve, concomitant with a decline in the degree of dispersion of parasites in the older age classes of hosts, can provide evidence of parasite-induced host mortality, the changes in mean abundance and over-dispersion of metacercarial stages of Diplostomum spathaceum, D. gasterostei, Tylodelphys clavata and T. podicipina in relation to fish age were studied in a field locality. The mean parasite burden of D. spathaceum, D. gasterostei and T. clavata increased with host age and the maximum mean burden was found in the oldest hosts. The variance to mean ratio also increased in D. gasterostei, but decreased in the oldest hosts in D. spathaceum and T. clavata. It is concluded that this decrease could be due to parasite-induced host mortality but could equally be due to death of parasites within the host or to changes in infection rate or could be a reflection of the small sample size of the oldest fish. The mean burden of T. podicipina declined gradually with host age, but the variance to mean ratio remained constant and it is concluded that this could be explained by death of the parasites within the host. None of these data or data from other localities provided clear and unambiguous evidence of host mortality induced by heavy infections of any of the four species, although they are consistent with such mortality and do not refute such a possibility. It is concluded that it may be just as difficult to detect and unequivocally demonstrate parasite-induced host mortality in metacercarial digenean–fish host systems as in any other parasite–host systems.

Macrogametogony and the formation of the oocyst wall has been examined in Eimeria acervulina. Macrogametes develop within a parasitophorous vacuole. Within the cytoplasm can be observed wall-forming bodies of Type I (WFBI). and Type II (WFBII), a nucleus, mitochondria, canaliculi and polysaccharide granules. WFBII are unusual in possessing a membrane internal to the granular endoplasmic reticulum. Formation of the outer layer of the oocyst wall is preceded by the separation of a veil membrane, and accompanied by morphological changes in the WFBI. WFBI material is deposited between the veil-forming membrane and the two cytoplasmic membranes. A newly formed membrane divides this outer layer during its early development. The inner layer of the oocyst wall is formed from WFBII material which is deposited between the cytoplasmic limiting membranes. The outer layer of the oocyst wall consists of granular and osmiophilic parts, but the inner layer of the wall is homogenous.

Temperature-dependent survival and reproduction of Gyrodactylus bullatarudis Turnbull, 1956 were examined at 17, 21, 25·5, 27·5 and 30 °C on isolated guppies under conditions where parasite density dependence and host responses were minimized. Parasites had the longest life-span (5·5 days) at 21 °C, the highest average fecundity (1·73 offspring) at 25·5 °C and the highest instantaneous per capita birth rate (0·543/parasite/day) at 27·5 °C. The intrinsic rate of increase of the parasite population was maximum (0·230/parasite/day) at 27·5 °C. Data obtained from this study indicate that G. bullatarudis are not able to survive at 30 °C.

Mice pre-treated with Corynebacterium parvum and later challenged with Plasmodium vinckei become infected but do not die whereas control mice do. When pre-treated mice were challenged with 1, 10, 1 × 102, 1 × 104, 1 × 105 or 1 × 106 parasites, the pre-patent periods correlated directly with the number of parasites injected, but the subsequent parasitaemias reached similar levels. This suggests that parasite killing, resulting from pre-treatment with C. parvum, is not triggered until the parasite load has reached a particular threshold. The injection of alloxan monohydrate, which brings about the release of toxic oxygen inter mediates thought to be involved in non-specific immunity, has little effect on P. vinckei infections until the parasitaemia is relatively high. This indicates that oxygen-mediated parasite killing also does not occur until the parasitaemia has reached a particular threshold. It is suggested that it is only at relatively high parasitaemias that the factors involved in parasite killing are able to enter the infected red blood cells.

The development of resistance by the Houghton strain of Eimeria tenella (H) to monensin has been studied by serially passaging the strain in groups of 30 birds, each inoculated with 106 oocysts and given 100 p.p.m. of drug in the food. After 16 passages a line partially resistant to this concentration of monensin was produced. Passage of the line in birds medicated with 200 or 300 p.p.m. of monensin did not result in the acquisition of a greater degree of resistance. A further reduction in sensitivity to 100 p.p.m. monensin was obtained, however, when the line was serially passaged in 15 groups of 6 birds each given 3 × 106 oocysts. Five passages in the absence of monensin resulted in restoration of sensitivity, suggesting that the resistance developed was not stable. The pathogenic effects of the line that had been passaged in chickens given 300 p.p.m. of monensin were less than those of the parent line.

The sheath and cuticle of microfilariae of Brugia pahangi were examined by electron microscopy and the presence of various proteins, carbohydrate and enzymes sought. The epicuticle of microfilariae consists of a pentalaminate structure (24·0 ± 1·4 nm), a cortex (13·7 ± 3·6 nm) and a basal zone (27·8 ±4·8 nm) which is often banded in appearance. The pentalaminate layers are not continuous at the base of the interannular grooves. The sheath and the epicuticle of B. pahangi stained positively with concanavalin A and saccharated iron oxide. The sheath of approximately 50% of microfilariae showed activity for acid phosphatase, 5′ nucleotidase and peroxidase, but not for ATPases, alkaline phosphatase or esterase. No enzymes were detected in the epicuticle although the cortex and basal layers of the cuticle did show enzymic activity. Structures beneath the cuticle in the main body of the worms contained considerable enzymic activity. Microfilariae directly isolated from the blood of infected cats were found by immunochemical means to carry serum proteins on their sheaths but not on their cuticles. These studies extend the definition of the outer structures of microfilariae and confirm that they significantly differ in morphology and enzyme content from typical mammalian cell membranes.

The filarial worm Litomosoides carinii showed a rapid uptake of glucose during in vitro incubation. This uptake proceeded linearly with time, and was significantly higher under aerobic compared to anoxic conditions. Under an atmosphere of nitrogen the worms converted glucose almost quantitatively to lactate, whereas in the presence of oxygen appreciable quantities of acetate, acetoin and CO2, in addition to lactate, were formed. Although aerobically only 73% of the carbohydrate carbon could be accounted for by the latter products as well as by a net glycogen synthesis, attempts to identify other compounds presumed to be derived from glucose metabolism have been unsuccessful. The complete sequence of the glycolytic enzymes was detected in particulate-free cytosolic extracts of the filarial worm. With the exception of 6-phosphofructokinase, all glycolytic enzyme activities were considerably higher than those reported for rat liver. In addition, L. carinii possesses the entire set of enzymes catalysing the eight successive reaction steps of the tricarboxylic acid cycle. On a mitochondrial protein basis, the specific activities of these enzymes were similar to those present in rat liver. Various enzymatic activities of the mitochondrial respiratory chain were detected in the parasite. These include low levels of NADH and cytochrome c oxidases, but a high activity value for NADH dehydrogenase. Cell-free extracts and the mitochondrial fraction of the worms were found to exhibit an enzyme capable of catalysing the decarboxylation of pyruvate. Since this activity was stimulated 5- to 20-fold by the cofactors known to be required by the pyruvate dehydrogenase complex of other animal cells, pyruvate decarboxylation and thus acetate formation in the parasite may be mediated by an enzyme similar to, or identical with, the pyruvate dehydrogenase system. Isotopic carbon balance studies and experiments in which substrates specifically labelled with 14C were employed showed that substrate carbon can to some extent enter into respiratory CO2. From these and the enzymatic analyses it is suggested that complete oxidation of carbon substrate may be of relevance as an energy-conserving pathway in the filarial worm.

Antigens of three lines of the rodent malaria parasite Plasmodium yoelii have been studied using crossed immunoelectrophoresis. P. y. yoelii line A1 is a mild line which is restricted to reticulocytes. P. y. yoelii line YM and P. y. nigeriensis line D1 are virulent infections which multiply in both immature and mature erythrocytes. One antigen, designated Py-1, was found to differ in its electrophoretic mobility between the lines, being fast (F) in lines A1 and YM and slow (S) in line D1. Antigen Py-1 also varied in quantity among the three lines; greater amounts were detected in parasites inhabiting mature erythrocytes than in those in reticulocytes. These characters were stable during blood and mosquito passage.

The inheritance of an antigen designated Py-1 in the rodent malaria parasite Plasmodium yoelii has been investigated. A cross was made between 2 lines differing in the electrophoretic mobility and quantity of Py-1 detected by crossed immunoelectrophoresis. In 10 clones isolated from the progeny of the cross the level of Py-1 always correlated with the virulence of the infection and it was concluded that these characters were different phenotypic effects of the same gene mutation. The electrophoretic mobility of Py-1 segregated independently of the virulence character and was therefore controlled by a different gene. These two antigenic markers also recombined with isoenzyme and drug-sensitivity characters distinguishing the parent lines.

In vitro studies of the behaviour of the trypanosomatid flagellates Trypanosoma brucei and Leishmania hertigi in the presence of cell-free haemolymph of locusts, Schistocerca gregaria and cockroaches, Periplaneta americana revealed the presence of parasite agglutinins. The range of normal values of agglutination titres was 2−4 to 2−13. Physico-chemical treatment of haemolymph indicated that these agglutinins are protein or glycoprotein in nature and are only partially affected by heat treatment below 65°C, at which temperature incubation of haemolymph for 30 min abrogated all agglutination. Agglutination was not dependent on the presence of Ca2+ or Mg2+. Prior infection of locusts and cockroaches with T. brucei and L. hertigi significantly increased agglutinin titres between Days 4 and 6 in cockroaches (P < 0·05) and from Days 2 to 4 when L. hertigi was inoculated into locusts. The induced differences in titres observed in locusts infected with T. brucei were not significant. Lysozyme levels were significantly increased after inoculation of T. brucei into cockroaches compared with placebo-inoculated and uninoculated controls. L. hertigi inoculation produced significant increases in lysozyme levels compared with controls between Days 1 and 7 in locusts and 3 to 6 in cockroaches. These studies indicate that, at least in easily manipulated model systems, induced responses to intrahaemocoelic inoculation to trypanosomes and Leishmania can occur. As far as we are aware this is the first report of an induced response of an insect to such important parasites. The possibility that induced responses in natural vectors to these parasites occurs requires investigation.

Haemolymph free amino acids (HFAAs) in adult Tenebrio molitor varied in concentration with beetle age. Between 9 and 30 days post-emergence the concentration range was 34–94 mM with a peak occurring in 15-day-old insects. Much of this age-related variation was due to changes in the concentration of a single amino acid, proline. An analysis of concentrations of individual HFAAs in 15-day-old beetles, following infection with metacestodes of Hymenolepis diminuta, revealed that changes in female insects are more marked than those occurring in males. In the former, changes occurred in the concentration of 9 amino acids (isoleucine, leucine, arginine, serine, threonine, tyrosine, phenylalanine, proline and alanine/citrulline) whereas in the latter only 4 amino acids were affected (threonine, glycine, histidine and arginine). It is suggested that H. diminuta may interact with the endocrine system of its beetle host.

The germinal layer of sterile 9-month-old murine peritoneal cysts of Echinococcus granulosus equinus shows interrelated variation in depth, tissue integrity, metabolic reserves and the number of autophagic lamellar bodies present. These features are similar in large and medium-sized cysts from the same host, whether occurring singly or within cyst masses. Deep germinal layers (> 16 μm) are lipid- and glycogen-rich and possess numerous autophagic vacuoles with 6 nm period lamellar stacks asymmetrically disposed peripherally; shallow layers (< 12 μm), with indications of degeneration, have depleted metabolic reserves and fewer lamellar bodies. These bodies are formed by smooth endoplasmic reticulum encirclement of small glycogen masses followed by further sequestration, and eventually definition of glycogen particles may be lost. Autophagy of mitochondria and cytoplasmic vesicles also occurs. The presence of lysosomal enzymes within the layer suggests autolysosomal compartmentalization of excess substrate and effete material. Mucopolysaccharide bodies, containing material similar to that exocytosed to form the laminated layer matrix, occur and are formed from fusion and autophagy of Golgi-derived vesicles. These bodies may also develop peripheral 6 nm period lamellar stacks, but of limited depth. Mucopolysaccharide bodies are the dominant feature of the germinal layer of very small cyst-mass cysts in which laminated layer production is considered to be arrested. They thus represent a repository for the unreleased mucopolysaccharide material.

The effects of metacestodes of Hymenolepis diminuta on haemolymph proteins of Tenebrio molitor are restricted to female hosts. In beetles aged 15 days post-emergence, and harbouring 12-day-old metacestodes, haemolymph concentration is 46·7% higher than in non-infected animals and similar effects are found in longer standing infections. Electrophoresis of haemolymph revealed the presence of 13 bands. Densitometric analysis showed that only band 2/3 was significantly elevated in infected hosts although band 7/8 also showed an increase. These bands were also present in egg homogenates and are thought to be vitellogenins. It is therefore proposed that the excess protein found in infected beetles is a female-specific protein resulting from an interaction between the parasite and the host endocrine system.

SDS-PAGE analysis revealed that the protein composition of mechanically transformed schistosomula (ms) of a Japanese strain of Schistosoma japonicum was essentially similar to that of a Philippine strain of S. japonicum. However, the protein components of S. mansoni ms were remarkably different from those of S. japonicum. Specific proteins of Mr 60–65.and 30 kDa were found in ms of S. japonicum and S. mansoni, respectively. In skin-penetrated schistosomula (ss) of the two species, a common protein of 67 kDa was detected. Using two-dimensional electrophoresis, strain-specific polypeptides (9 in a Japanese and 11 in a Philippine strain) were identified in ms of S. japonicum. Mechanically transformed and skin-penetrated schistosomula of S. japonicum (Japanese) had, respectively, 7 and 10 specific proteins. Four and 1 specific polypeptides were also detected in two-dimensional profiles of ms and ss of S. mansoni, respectively.

The immune-dependent elimination of a challenge infection in rats vaccinated with highly-irradiated cercarriae of Schistosoma mansoni was analysed by passive transfer of serum, recovery of the challenge from the lungs and liver and by transferring lung-stage schistosomula. Recipients of serum from rats immunized with either unirradiated, 20 or 40 krad.irradiated cercariae, most of which die in the liver, lungs and skin, respectively, were equally resistant if the serum was injected on the day of infection or 5–7 days after infection. In addition, vaccinated rat serum transferred to mice and vaccinated rabbit serum transferred to rats conferred comparable protection when injected on day 0 or 5 days after infection of the recipents. This apparent susceptibility of the lung schistosomula to immune attack was confirmed by challenging 20 or 40 krad.-irradiated cercariae vaccinated rats with lung-stage schistosomula derived either from mice or rats. All the detectable attrition of a cercarial challenge in vaccinated rats occurred between 7 and 10 days post-challenge, before the parasites reached the liver. Since there was no evidence of damage or attrition in the skin or lungs before day 7 it was concluted that immune-dependent elimination occurred rapidly following a ‘window of sensitivity’ coinciding with the migration of the parasites from the lungs to the liver.

The development of the egg-shell of the trichuroid nematode, Trichuris muris, was observed by light and electron microscopy. Initial stages of shell formation occurred immediately following sperm entry into the oocyte in the spermatheca of the female worm. The external vitelline layer separated from the oolemma, reticulate oocyte granules were discharged into the resultant perivitelline space and stored glycogen contributed to the formation of the chitinous shell. A lipid layer developed between the inner surface of the chitinous layer and the oolemma. The fully formed egg-shell thus comprised an external vitelline layer, a middle chitinous layer and an inner lipid layer. The outer vitelline layer resembled a unit membrane with strands of particulate material attached to its outer surface. The middle chitinous layer was composed of chitin microfibrils encased in a protein coat; the sheets of microfibrils were arranged as parallel lamellae forming a helicoidal architecture. The inner lipid layer contained a variable number of loosely arranged strands of electron-opaque and less-opaque material.

Studies of antigenic variation in Trypanosoma vivax have been hindered by its low infectivity to laboratory hosts and difficulties in obtaining defined cloned populations. The recent isolation of mouse-infective T. vivax has partially overcome the first problem. We have developed a highly efficient cloning technique which has been applied to the isolation of a collection of 71 clones, representing 31 distinct variable antigen types, of mouse-infective T. vivax from Nigeria. This serodeme showed no cross-reaction with several T. brucei-group serodemes, but some cross-reaction with naturally occurring Kenyan T. vivax.

The larval output of Angiostrongylus cantonensis at different initial densities in rats was examined throughout the reproductive life-span of the worms using a modified dilution technique. With an initial density of 2 worms the mean duration of larval output was 414·2 days. The output was at a level above 50000 larvae/female/day from 15 to 45 weeks after infection. The mean total output was 18·9×106 larvae/female. The larval output/female decreased with an increase in the worm burden and also with the passage of time after infection. The longevity of host rats also decreased with increasing worm density and many of the heavily infected rats died early during infection. These results suggest that worm density has an effect on the fecundity of this parasite, and that the effects may be due to fibrous changes in host lung tissue but not changes in the physiology of the worms. It is also suggested that density-dependent effects on fecundity play a role in the population dynamics of both the parasite and host as regulatory mechanisms in the field.

Intact surface membrane vesicles were obtained and purified from the schistosomula of Schistosoma mansoni. The method of preparation of the vesicles involved treating the schistosomula in a buffered high salt medium for 1 h at 4°C (Step 1), and then for 1 h at 25–37°C (Step 2). Vesicles were formed from mechanically- and skin-transformed schistosomula, and their size and number depended on the temperature of Step 2. The majority of the vesicles expressed surface membrane concanavalin A receptors and parasite antigens on their outermost surfaces. When incubated with sensitized peripheral blood mononuclear cell preparations from humans exposed to schistosomal preparations, the vesicles stimulated lymphocyte transformation as effectively as soluble egg and adult worm antigens. Although both preparations contained identical proteins, small vesicles were less effective than large vesicles in stimulating lymphocyte transformation. Hence, vesicles prepared under a variety of conditions might be used to study those factors which influence the presentation of membrane-bound surface antigens to the immune system of the host.