Electrophoretic variation of the enzymes glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase (NADP-dependent) has been studied in the African murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi and their subspecies. Horizontal starch gel electrophoresis was used throughout. The number of isolates examined in each subspecies varied from 1 (P. y. nigeriensis) to 24 (P. c. chabaudi). Extensive enzyme variation was found among isolates of most of the subspecies from which more than two such isolates were available for study. It is clear that the phenomenon of enzyme polymorphism is of common occurrence among malaria parasites. With the exception of P. berghei and P. yoelii, of which all isolates share an identical electrophoretic form of lactate dehydrogenase, no enzyme forms are shared between any of the 4 species of murine plasmodia. By contrast, within each species common enzyme forms are shared among each of the subspecies. The subspecies are, nevertheless, distinguished from each other by the electrophoretic forms of at least one enzyme.

The distribution and reassortment of enzyme variation among isolates of a single subspecies is in accordance with the concept of malaria parasites as sexually reproducing organisms. The study of variation among parasites present in individual wild-caught rodent hosts demonstrates that natural malarial infections usually comprise genetically heterogeneous populations of parasites. Nevertheless, the number of genetically distinct types of parasite of any one species present in a single infected host appears to be small. Generally not more than 2 or 3 clones of parasite of distinct genetic constitution are present in a single infected animal.

The succinate dehydrogenase of adult Fasciola hepatica was found to exist in active and inactive forms. The enzyme was inactivated by 1 μM oxaloacetate and activated by incubation with compounds which bind to the active site (succinate, fumarate, malonate) or by incubation with anions and certain nucleotides. The activation of the enzyme by succinate followed first-order kinetics. The extent of activation of F. hepatica succinate dehydrogenase depended on the nature and concentration of the activator and on the pH. The rate of activation of the enzyme depended on the temperature. In contrast, the fumarate reductase activity of F. hepatica was not activated by incubation with substrate or anions and was not inhibited by oxaloacetate (100 μM). The significance of these results in the regulation of the tricarboxylic acid cycle in parasitic helminths is discussed.

Sheep infected with adult Fasciola hepatica were drenched with rafoxanide. At 4, 8, 16 and 24 h after drenching the sheep were killed and the flukes removed, washed and rapidly frozen in liquid nitrogen. The content of key metabolites in the fermentation pathway were determined and compared with those in control F. hepatica, whose hosts were not treated with rafoxanide.

Rafoxanide decreased glycogen, malate, NADH and ATP levels. The level of other metabolites in the pathway increased for the first 8–16 h after rafoxanide treatment. The marked decrease in ATP and glycogen, and the increase in total [NAD+]/[NADH] and [oxaloacetate]/[malate], together with the changed content of other metabolites, led to the conclusion that the mode of action of rafoxanide against F. hepatica in vivo is by uncoupling oxidative phosphorylation.

Rats were treated with betamethasone at different stages of infection with Strongyloides ratti. Treatment caused a diminution in the number of adult worms recovered but this residual population was not expelled as were worms from untreated rats. If treatment was started after expulsion had already begun adult worm numbers increased – suggesting that auto-infection occurred. Treatment throughout a primary infection prevented the development of an immune response which expelled, or killed, a challenge infection. Treatment, just before and just after challenge, prevented the expulsion of the challenge infection. Worm expulsion up to day 35 was prevented by treating rats with betamethasone 4, 6, 8 and 10 days after infection.

The cytotoxicity of normal rat spleen cells to antibody-coated Trypanosoma cruzi epimastigotes has been studied by assaying the release of [3H]-labelled macromolecules from the parasites. The release of thymidine (DNA) is slower than the release of uridine (RNA), suggesting that the nucleus is broken down more slowly than the cytoplasmic membrane. Less than 50% of the leucine (protein) is released when the parasites are lysed, whereas uridine (RNA) is almost totally released. In practical terms these results show that the release of incorporated radioisotope-labelled uridine can be used as a sensitive assay for cytotoxicity of T. cruzi. Cytotoxicity by normal rat spleen cells is antibody dependent and proportional to the logarithm of effector cell number. The lag phase and the rate of RNA release is not altered by centrifuging the parasites and effector cells to enhance contacts between them.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. rangeli and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconstituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools.

The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.

Fresh (3 h) schistosomula of Schistosoma mansoni were obtained by the skin penetration method and injected intravenously into groups of mice either immediately or after 48 h incubation in tissue culture. Schistosomula were then recovered from the lungs 1 to 2 days after injection. The recovery results showed that the ability of uncultured schistosomula to emerge from lung tissue increased with time, became maximal on the 3rd day and diminished thereafter. In contrast, cultured schistosomula were able to emerge from chopped lung at a much earlier time after injection.

Fresh schistosomula, as well as those recovered from mouse lungs, were assayed by indirect immunofluorescence for the presence of parasite and host antigens. Similarly, fresh schistosomula which had been incubated in foetal calf serum, with or without mouse red blood cells, were assayed for parasite and host antigens after 3–24 h of culture. Fresh schistosomula expressed parasite antigen with greater intensity than those recovered from mouse lungs and showed no trace of host antigen. Lung schistosomula, on the other hand, expressed less parasite antigen but more red blood cell antigen with increasing time in the host. Schistosomula cultured in the absence of mouse red blood cells expressed only parasite antigen throughout the period in culture, while those cultured in the presence of mouse red blood cells also expressed parasite antigen during the entire period in culture but, in addition, by 24 h host antigen could also be detected.

These developmental and adaptational changes may play a role in determining the survival of parasites in the normal or immune host.

The freeze fracture technique has been used to quantify changes in the integral components of the double outer membrane of Schistosoma mansoni during the 6-week period of development within the mouse. The intramembraneous particle (IMP) density on the P1 face begins to rise Within 6 h of host penetration, reaches a maximum at day 4 and then falls rapidly after day 9, so that it is at a low level between 3 and 6 weeks. The E1 face IMP density follows the same course as that of the P1 face except that maximum particle density is recorded on day 1 and the counts begin to fall on day 5. The IMP density on the P1 face remains at a consistently low level throughout development. The E2 face IMP density rises gradually to a peak at day 4, when the parasites have migrated to the lungs, and remains thereafter at a similar level, so that by 6 weeks the E2 face has a higher IMP density than the other three fracture faces. The E2 face IMP show a marked increase in size on day 4. Morphological studies indicate that a different type of inclusion body makes a transient appearance in the tegument of the lung worms, and immunocytochemical techniques show the lung worms to be nonimmunogenic. It is suggested, therefore, that the E2 face IMP may represent complexes of parasite antigens and acquired host antigens. The tegumental membranes of cultured specimens have also been examined by freeze fracturing and the IMP densities compared with those obtained from in vivo parasites; the cultured schistosomula have a lower E2 face particle density than the in vivo specimens.

Changes in the ultrastructure of the daughter sporocyst and contained metacercariae of Microphallus pygmaeus during culture in artificial seawater and modified Medium 199 were examined and compared with variations in oxygen consumption. The very early commencement and rapid progression of autolysis in the sporocyst body wall as compared with little or no change in most metacercariae in both media, together with the elevated oxygen consumption in the nutrient medium, suggests that most of the oxygen is consumed by the contained metacercariae. The almost simultaneous onset of body wall degeneration in both media suggests that the nutrient medium is not suitable for the maintenance of healthy daughter sporocysts.

In the fully formed daughter sporocyst of Microphallus similis (Jäg.) the tegument is divided into a wide, outer cellular epithelium and a narrow, inner electron-dense anucleated microvillous syncytium. The narrow subtegument consists of circular and longitudinal muscle, nervous, synthesizing and germinal cells. It is suggested that during development of the tegument, an undifferentiated nucleated syncytium becomes divided by the formation of membranes between the nuclei and between the narrow, inner electron-dense microvillous and the outer cellular regions. The outer nucleated region of the tegument is homologous to the nucleated investing syncytium which surrounds redial and cercarial embryos and which is shed later in development. The retention of the nucleated region in some daughter sporocysts is, therefore, an example of paedogenesis.

Synergy between clopidol and methyl benzoquate against Eimeria maxima was shown to be supra-additive. Collateral sensitivity to these drugs could not be demonstrated in resistant lines of this parasite. Resistance to methyl benzoquate and clopidol was not transferred when lines of E. maxima, resistant to the respective drugs, were propagated together. The failure to demonstrate this phenomenon was judged not to be due to synergy between the drugs. Attempts to induce simultaneous resistance to both drugs failed, although resistance to methyl benzoquate was readily acquired by a line of E. maxima resistant to clopidol. Induced resistance to clopidol in a methyl benzoquate-resistant line required numerous passages.

Larvae of Boophilus microplus were labelled with [32P] and used to study the rejection of larvae from British breed cattle with different levels of resistance. On animals of high resistance the loss of larvae due to grooming ranged from 9–54% during the first 24 h of infestation, and more time was spent grooming by these animals. Most of the grooming activity could be attributed to the presence of tick larvae and the presence of older tick stages did not increase the percentage of larvae lost. Grooming was directed to attached larvae and these could be removed. Animals of low resistance did not lose a significant number of larvae as a result of grooming, but all previously infested hosts lost a proportion of the larvae (18–39%) which could not be accounted for by grooming. This loss was greater than the total loss of larvae (11%) on animals not previously exposed to B. microplus.