The purpose of this workshop was to collect together colleagues investigating the intermediary metabolism of protozoa, with a view to discussing those pathways involved in energy metabolism and the production of ATP and other high-energy compounds, together with the factors affecting energy balance. The aspects of energy metabolism chosen for discussion comprised the metabolic pathways ranging from the strictly anaerobic to highly oxidative; subcellular compartmentation of these pathways within the protozoa; the functional role of these pathways including a consideration of aero-tolerance; and the use of inhibitors as biochemical probes and potential chemotherapeuticagents. Hopefully this approach has produced a broad 'over-view' of important areas of protozoan energy metabolism which will enable both the specialist and non-specialist to appreciate the similarities and differences between the metabolic behaviour of a range of protozoa.

Cultured adult chick kidney (CK) cells inoculated with sporozoites of Eimeria tenella showed progressive alterations in their morphological and adhesive characteristics. Scanning electron microscopy (SEM) showed that during a 3-day period following the inoculation of parasites there was a gradual loss of contact between the cells. Initially, the cells remained connected by long cytoplasmic bridges but with the breakdown of these, the cells began to round up, losing complete contact with each other and remaining attached to the substratum by means of retraction fibres. By 3 days many cells had completely detached. The factors inducing these alterations were transmissable via the medium as the alterations were mimicked by non-parasitized cells in co-culture with parasitized cells. Studies with the reflection interference microscope (RIM) showed that the changes were accompanied by an increase in the cell-substrate separation distance and the loss of focal contacts. These changes were not effected by substances transmissable via the medium. Parasitized cells showed enhanced agglutination with Concanavalin A (Con A) which could be eliminated by pre-fixation. The possibility that changes to the host cell indicate a rearrangement of cytoskeletal apparatus is discussed.

A rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 × 1010 untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90 % of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Freshly excysted Fasciola hepatica possess an outer glycocalyx which stimulates an antibody response in cattle infected with F. hepatica metacercariae. Sera from animals receiving either a single or a double infection were examined for levels of IgM, IgG1 and IgG2 specific for the outer glycocalyx of intact live juvenile F. hepatica. High levels of specific IgG1 were found to predominate. Using positive sera in an indirect fluorescent antibody assay, the bovine immunoglobulins labelled the outer glycocalyx in a characteristic reticulated pattern. On incubation at 37 °C this pattern rapidly broke up into discrete clumps and progressed until the entire antibody–outer glycocalyx complex was shed. The shedding phenomenon was shown to be continuous and, by using preparations of Fab fragments, was not due to the bivalent binding of the sensitizing antibody.

Schistosomula of Schistosoma mansoni, recovered either after penetration of cercariae through isolated rat skin or by mechanical transformation of cercariae, become fully resistant after 24–48 h of culture to damage by human blood eosinophils in the presence of human anti-schistosomular sera. Cultured schistosomula are also shown to lose their susceptibility to attack by human eosinophils in the presence of human complement. This resistance is related to the simultaneous reduction of the ability of human anti-schistosomular antibodies and human complement component C3 to bind to the surface of the cultured larvae. The development of insusceptibility to antibody, complement and eosinophil-mediated attack does not require the acquisition by the schistosomula of a protective coat of host or other macromolecules, since it is achieved in chemically defined culture medium free of serum and macromolecules. This supports the hypothesis that schistosomula undergo intrinsic changes which render them insusceptible to immune attack.

Even if we do not always acknowledge the fact, nutrition is a fundamental aspect of symbiosis and parasitism. (1) The significance of nutrition often forms a basic feature in schemos of classification of the various associations between living organisms (see Read, 1908; Whitfield, 1979). (2) The growth and reproduction of different parasites appear to Vary in response to the nutrition of the host (see Mettrick & Podesta, 1974; Nesheim, Crompton, Arnold & Barnard, 1977; 1978; Parshad, Crompton & Nesheim, 1980). (3) The course of a parasitic infection may also vary in response to the nutritional status of the host and perhaps such variations are the result of the affocts of nutrition on the host's immune response (Chandra & Nowberne, 1977; Chandra, 1980). (4) Finally, parasitic disease is well known to be much more scrious in undernourished hosts (see Chandra & Newberne, 1977); when man's the deprived host, the impact of infections and parasitic disease on young children is Particularly severe and distressing (Latliam, 1975).

Neither phosphagens nor phosphagen phosphotransferase activity could be detected in Fasciola hepatica, Hymenolepis diminuta, Moniezia expansa or in the plerocercoids of Ligula intestinalis. The plerocercoids of Schistocephalus solidus, however, possess an active taurocyamine phosphotransferase, although it too contains no detectable phosphagens. The taurocyamine phosphotransferase of S. solidus has an absolute requirement for a divalent metal ion and ATP could not be replaced by ITP, GTP, CTP, or UTP as the phosphate donor. The role of phosphagens in helminths is discussed.

The pattern of DNA synthesis in chicken kidney (CK) cells parasitized by Eimeria tenella was altered; a 2-fold increase in the uptake of [3H]-thymidine occurred in these cells compared with that shown by the controls. This difference was significantly different at 16 h post-inoculation (p.i.) and reached a peak at 20 h p.i. Non-parasitized cells in co-culture with parasitized cells showed a level of incorporation intermediate between that of the parasitized and non-parasitized mono- cultures. It was shown that this stimulation was due to a factor released into the medium by the parasite or the parasitized cell. A possible course of events leading to induced and prolonged DNA synthesis by the host cell and the significance of this change in the life-cycle of the parasite is discussed.

Adult Ostertagia circumcincta from freshly killed sheep were incubated at 39 °C in a medium containing inorganic salts, antibiotics and D-[U-14C] glucose. The worms appeared healthy even after incubation for as long as 72 h. All the radioactivity was recovered either within the worms or in the incubation vessel in the form of metabolic products or unmetabolized glucose. Incubations were carried out at low oxygen tension except for those in which CO2 was measured. These were either aerobic or anaerobic. In terms of both quantity and radioactivity the main metabolic products of glucose were CO2, propan-1-ol, acetate and propionate. Smaller amounts of ethanol, lactate and succinate were formed. The results are compared with those found for the similar nematode Haemonchus contortus.

The ultrastructure of immature exo-erythrocytic forms of Plasmodium berghei in rat hepatocytes was studied at stages between 25 and 51 h of development. A new method was successfully applied to localize the parasites in a small portion of the liver by temporary ligature of blood vessels to the majority of the liver, and from the spleen and the pancreas. Nuclear profiles appeared to be part of a highly lobed nuclear reticulum. Peripheral vesiculation and vacuolization of the cytoplasm was increasingly prominent and ushered in the formation of pseudocytomeres. Spacing between host- and parasite-derived membranes could first be observed after 43 h of development. In general, organellar development was found to follow closely that described for plasmodial oocysts.

Although methods for quantitative immunoassay have been available for a long time and have been used as a diagnostic tool for viral and bacterial diseases, as well as for immunological disorders, it is only recently that such methods have found wider application in parasitology. There are probably several reasons for this. One is that in developed countries parasitic diseases and in particular tropical infections were for a long time not recognized as severe health problems which deserved serious attention. Many technical problems have also been associated with parasite serology. Earlier serological methods were, for various reasons, unsuitable for use in the diagnosis of parasitic infections. For instance, the complement fixation test is, in many instances, not sensitive enough to detect active infection and the later introduced passive haemagglutination test, which has a considerably higher level of sensitivity, has the disadvantage of not detecting antibodies to carbohydrate antigens, such as some of the diagnostic antigens of adult schistosomes.

Three types of vaccine (1) a sonicated extract of killed larvae, (2) 5-fluorouracil-treated larvae and (3) 40 krad. X-irradiated larvae have been evaluated. Pups aged 3–4 months, weighing 2± 0·5 kg and free of natural hookworm infection were selected for experimental studies. The sonicated extract was given either in a single dose or as 2 doses at weekly intervals, whereas the 5-fluorouracil-treated and X-irradiated larvae were given in a single dose only. After completion of the vaccination schedule, the pups were challenged with filariform larvae and were sacrificed 3 weeks after challenge. Non-vaccinated pups and those vaccinated with a single dose of sonicated extract lost weight and had diarrhoea or dysentery after challenge. Furthermore, in non-vaccinated pups, haemoglobin levels fell and at autopsy the intestinal mucosa was found to be grossly congested and free blood was present in the lumen of the intestine. Such findings were not obtained in pups vaccinated with the other types of vaccines. The percentage of infective larvae which developed to the adult stage in non-vaccinated pups was 82·7 ± 9·53 compared with 45·1 ± 12·33, 30·1 ± 10·05, 20·6 ± 10·60 and 8·0 ± 7·46 %, respectively in pups vaccinated with a single dose of sonicated extract, double dose of sonicated extract, 5- fluorouracil-treated and 40 krad. X-irradiated larval vaccines. While the irradiated larval vaccine provided maximum protection against clinical ancylostomiasis, 2 doses of the sonicated larval vaccine could also afford almost equal protection to pups.

The daily food intake and related changes in body weight were measured in young rats which were fed on synthetic diets containing 1% (3 experiments), 2% (4 experiments) and 20% (1 experiment) casein respectively. Some rats were infected with Nippostrongylus brasiliensis and allowed to feed ad libitum, some remained uninfected and were allowed to feed ad libitum while others, which were matched by initial weight with rats from the infected group, remained uninfected and were given only the same amount of food as that consumed during the previous 24 h by their infected partners. No significant differences were detected between the rats before the start of the infection. Thereafter, the food intake of the infected undernourished rats was usually found to be significantly lower and the loss of body weight significantly greater than those of the uninfected rats. In infection periods of relatively long duration, the pair-fed uninfected rats did not lose as much weight as their infected partners even though their food intake (1% and 2% casein) was the same. Although the food intake, and consequently the weight, of infected rats fed on the diet containing 20% casein were significantly less than in their uninfected partners, no significant differences were observed between the weights of the infected and pair-fed rats fed on this diet.

The oncomiracidia larvae of monogeneans are small, ciliated, relatively slow-moving organisms with a limited free-swimming life. For example, the oncomiracidium of the skin parasite Entobdello, soleae is 250 µm in length, swims at a speed of 5 mm/sec and lives for about 24 h (Kearn, 1967a). Oncomiracidial survival is dependent on the larvae finding and attaching themselves to their specific fish hosts and selection pressure has been exerted on the timing of hatching of oncomiracidia and on their behaviour during their short freeswimming lives to enhance the chances of making this important contact.

Antibodies to Toxocara were measured by enzyme-linked immunosorbent assay (ELISA) in sera of ascarid-free dogs which had been fed 0,100 or 10000 embryonated T. canis eggs. Dogs fed 10000 eggs developed higher titres than those given 100 eggs and larvae failed to complete their migration to the gut. In addition, in dogs given 100 eggs, there was a significant negative correlation between the number of worms in the gut and granulomatous foci in the lung. The data suggest that the ELISA is sensitive and specific for Toxocara infection in dogs.

A strain of the cattle piroplasm Babesia divergens isolated from a fatal human infection was propagated in the Mongolian gerbil through 150 semi-continuous intraperitoneal passages. The infection was normally fatal; death, accompanied by profuse haemoglobinuria and debilitation, occurred as early as 44 h after intraperitoneal inoculation of heavily parasitized blood with precipitous drops in red blood cell and platelet counts. The average maximum parasitaemia achieved increased on continuous passage reaching 80% by the 150th stage. Twenty-four hours after infection erythrophagocytosis and splenic congestion were apparent by light and electron-microscopical examination and by 48 h hepatic necrosis, renal tubular damage with haemoglobin cast accumulation and ischaemic necrosis of ileal mucosa had developed. Gerbils were highly susceptible to small numbers of parasites when the inoculum was either fresh parasitized blood in high dilution or erythrocytes concentrated from animals showing minimal parasitaemia. Animals inoculated with parasites preserved in dimethyl sulphoxide at low temperatures usually developed fatal infections. However, occasionally animals suffered at most a low grade parasitaemia subsequent to recovery with parasite elimination. These animals were immune to further challenge, and no chronic infections developed. A. field strain of B. divergens isolated locally from a case of bovine redwater behaved similarly to the human strain on continuous passage in gerbils.

Histochemical and thin-layer chromatographic (t.l.c.) analyses were made on neutral lipids in the free (unencysted) metacercariae of Amblosoma suwaense (Brachylaimidae). As determined by t.l.c. the major neutral lipid fractions in metacercariae removed directly from Campeloma decisum snails were free sterols and sterol esters. Metacercariae incubated for 1 h at 37±1° C in sterile Locke's solution released mainly sterol esters and a lesser amount of free sterols into the medium. As determined by Oil Red O (ORO) staining, metacercariae accumulated neutral lipid in the intestinal caeca during incubation and the excretory system was ORO negative. Behavioural studies showed that metacereariae paired and aggregated in vitro and were attracted to lipophilic but not to hydrophilic worm products. Following t.l.c. preparative analysis it was demonstrated that metacercariae were attracted to sterol ester worm products but not to free sterol products.

Pasture plots in a warm coastal region were separately contaminated in the autumn, winter and spring with eggs of a coastal strain of Ostertagia ostertagi derived from dairy calves. At the same times, a parallel set of plots was contaminated by a strain transferred from within a beef cattle management system in a cooler tablelands region. A reciprocal transfer of the coastal strain was also made to the tablelands, where pasture plots were contaminated with eggs of both strains of O. ostertagi in winter. Successive groups of parasite-free calves were grazed on the plots for 10–14 days at 4-week intervals and then killed for worm counts 14 days after their removal from pasture. In both environments, irrespective of the season of pasture contamination, inhibition of development reached a maximum in spring and the two strains differed only in the proportion of the population inhibited, which was significantly greater for the table- lands strain. There was no strain difference for either numbers of infective larvae on pasture or total O. ostertagi numbers. This suggested that the difference between coastal and tablelands populations in the extent of inhibition is genetically determined. If an environmental stimulus was implicated in the spring occurrence of inhibition, the stimulus was provided equally in coastal and tablelands environments and it cannot be exposure to low temperatures.

Insecticide resistance is a serious problem in the control of many vector-borne diseases, particularly in developing countries where the possibility of changing to an alternative compound may be restricted. Consequently genes giving susceptibility to insecticides in vector species can be viewed as constituting a valuable resource to be used up with discretion. The exchange of information and thought taking place in the Workshop was centred upon possible ways to conserve this resource, i.e. to slow down the evolution of resistance while maintaining adequate control, so preserving the effective life of available insecticides as long as possible. The subject was treated mainly in relation to insect vectors of disease although niost of the discussion would also be relevant to other insect pests.

On day 10 of a primary infection of Trichinella spiralis in NIH mice, when adult worms were in the process of being expelled, their fecundity was markedly depressed. Upon transplantation to new host mice, the worms were able to establish successfully in their usual position in the anterior small intestine, even if originally recovered from the large intestine of mice expelling their worms, and these worms fully recovered their fecundity after 7 days in recipients. On day 5 of secondary and tertiary infections, immediately before loss of worms began, the adult worms were stunted and had a depressed fecundity. However, such worms, when transplanted, were able to establish in normal numbers and in a normal position in the intestine of recipient mice. Additionally, these worms were found to have developed a fecundity, and grown to a length, comparable to that of normal worms, but slightly less than that of control worms transplanted from day 7 of a primary infection, after 7 days in a new host. Electron microscopy showed that worms taken at the time of expulsion in primary and tertiary infections showed structural abnormalities, including large quantities of lipid in intestinal cells and somatic musculature and decreased quantities of stored glycogen. Upon transplantation to new host mice, these worms were found to have recovered a normal ultrastructural appearance within 7 days, although there was still evidence of a slightly elevated lipid and depressed glycogen content. It is concluded that immune expulsion of T. spiralis from mice does not involve irreversible damage to the worms.