Both asexual and sexual development of Plasmodium berghei was synchronized without chemical intervention using in vitro culture techniques. Combined in vivo and in vitro experiments were performed on the relationship between age, morphology and maturity of gametocytes. Schizogony took 22–23 h in the experiments. At 26 h post-invasion (p.i) the first males became capable of exflagellation. By 20 h p.i. the first gametocytes were recognizable in Giemsa-stained smears but the sex was hardly distinguishable until maturity (26 h p.i.). Survival time of gametocytes was estimated at 26 h in vitro (half-life 13 h) and the same survival time was suggested for gametocytes in vivo. Schizonts of P. berghei apparently disappeared from the peripheral circulation upon maturity, rupturing almost immediately. Mature schizonts in vitro persisted up to 48 h p.i. in non-agitated cultures. No evidence was collected for sequestration of any sub-population of gametocytes.

The sensitivities of promastigotes and amastigotes of Leishmania donovani to reagent or glucose oxidase-generated hydrogen peroxide (H2O2) were examined in a phagocyte-free system and compared with direct measurements of loss of H2O2 due to reaction with the parasite. Using a combined fluorescence dye uptake/dye exclusion viability assay in conjunction with motility and transformation data it was shown that log-phase promastigotes harvested from recently transformed cultures were intermediate in their H2O2 sensitivity between amastigotes and log-phase promastigotes harvested from long-term subcultures. It was also observed that, while promastigotes are equally sensitive to either form of H2O2 stress, amastigotes are more resistant to single larger amounts of reagent H2O2 than to equivalent amounts of H2O2 generated over a 1 h period. In each case the respective LD50 values obtained for each form of the parasite under each type of H2O2 stress correlated with saturation of their ability to remove H2O2 from the phagocyte-free system. For both promastigotes and amastigotes there was always a time delay after removal of either form of H2O2 stress before H2O2-mediated damage to membranes became apparent. The results suggest that the differential responses of promastigotes and amastigotes to different forms of H2O2 stress may depend upon different H2O2 scavenging mechanisms examined in more detail in the accompanying paper.

It was demonstrated that antimycotic agents are unsuitable for elimination of mycotic contamination in in vitro cultures of Leishmania spp. Ion-exchange chromatography was designed to separate the protozoan parasites from the contaminating yeast or fungal cells based on their surface net charges. The method was found to be efficient and rapid, and would also reduce bacterial contamination in cultures which have been exposed to both contaminants Viability of the cells has been shown to be unaltered by this treatment as demonstrated by their ability to multiply rapidly in culture medium, as was seen in the controls.

Different hydrogen peroxide (H2O2)-scavenging mechanisms, and the conditions under which they operate, have been examined in promastigotes and amastigotes of Leishmania donovani. For promastigotes, the ability of the parasite to remove H2O2 was completely ablated by sonication whereas for sonicated amastigotes substantial loss of H2O2 from the phagocyte-fre¸e test system still occurred. In direct contrast, the ability of amastigotes, but not promastigotes, to remove H2O2 was markedly inhibited by aminotriazole or sodium azide. This suggested a role for haem-containing enzymes, catalase or peroxidases, as a protective H2O2-scavenging mechanism and was consistent with detection of catalase in amastigotes but not promastigotes using a spectrophotometric assay. Both forms of the parasite did, however, show reduced ability to remove H2O2 at 5–7°C indicating that additional enzymatic scavenging mechanisms may operate. Glutathione peroxidase activity was undetectable in either form of the parasite. The total thiol sink, glutathione (GSH) plus protein thiols, was greater in promastigotes but the ability to regenerate GSH via glutathione reductase was equivalent for promastigotes and amastigotes. Less temperature-dependent non-enzymatic mechanisms (e.g. an unsaturated lipid sink) also appear to contribute to removal of H2O2 by both promastigotes and amastigotes. It seems likely, nevertheless, that the difference in H2O2 sensitivity between the two forms of the parasite relates to the activity of the direct H2O2-scavenging enzyme, catalase, which appears to operate more efficiently against a bolus of reagent H2O2.

A considerable proportion of mice lose acquired immunity to Plasmodium berghei during the first pregnancy. Immune parous mice, however, have a better immune status than virgin mice, the risk of loss of immunity during a subsequent pregnancy is greatly reduced, the capacity to clear parasites is enhanced, and the maintenance of immunity is less dependent on certain splenic functions. The establishment of improved immunity is dependent on the presence of proliferating parasites during the second half of pregnancy when immunosup pression results in recrudescence. Immune reactivity is also improved after a (chemothera peutically controlled) recrudescent infection provoked by immunosuppressive treatment of immune mice with corticoids or anti-T cell serum. This mimics the situation encountered during pregnancy. Hence, improved immunity after pregnancy is a consequence of a reconfrontation of a suppressed and/or convalescent immune system with proliferating parasites.

The morphology of Kronborgia pugettensis sp. nov., an endoparasite from the haemocoele of the caridean shrimp Heptacarpus kincaidi is described from specimens collected in the San Juan Island region of the western coast of North America. Members of this species are unisexual as are other members of the genus, but only females were found. An ootype and a type of accessory reproductive gland not known from other Kronborgia spp, are described, but they are probably present in other members of the genus. The life-history is similar to that of other Kronborgia spp. After the female reaches maturity, it emerges from the host and secretes a tubiform cocoon around itself and deposits its egg capsules within it. Embryogenesis lasts about 4 months. A free-swimming larva locates the new host, attaches to its exterior, and secretes a cyst around itself. It then penetrates through the exoskeleton to reach the haemocoele. K. pugettensis is easily distinguished from other members of the genus by the shape of the cocoon, type of host, and geographic distribution. This is the second species of fecampiid described from North America, but several undescribed species are known to exist from their cocoons.

Plasmodium berghei ookinete formation in vitro and within the midgut of susceptible Anopheles atroparvus were compared. No significant morphological differences were seen, except that in vitro development was more synchronized and less degenerating forms occurred. In vitro ookinete yields were 4–31 times higher and less variable than those in vivo. Mosquitoes of a susceptible and of a refractory line of A. atroparvus were simultaneously fed on the same host or via a membrane with the same suspension of in vitro-formed mature ookinetes. Up to 100% of mosquitoes of the susceptible line produced oocysts, mostly in high numbers, whereas infection rates and numbers of oocysts produced in mosquitoes of the refractory line were lower and much more so after host feeding than after membrane feeding of mature ookinetes, indicating that refractoriness does not depend on a single process of inhibition.

In vitro formation of Plasmodium berghei ookinetes was studied. Gametocytes produced in vitro were obtained from heart and tail blood of Swiss mice and from blood removed from mosquitoes directly after feeding on these mice. In vitro produced gametocytes were obtained from short-term cultures of the erythrocytic stages of P. berghei. Reproducible ookinete production was obtained in medium RPMI 1640, pH 7°8–8°0, using in vivo and in vitro produced gametocytes. The morphology of developmental stages of ookinetes and degenerate forms at the light microscope level is described. More ookinetes were produced in medium RPMI 1640 compared to MEM and ookinete yield – defined as the ratio between the number of in vitro produced ookinetes/105 erythrocytes and the number of exflagellations/105 erythrocytes in the infected blood – increased with lower erythrocyte densities in the cultures within the range of dilutions tested. A linear relationship existed between gametocytaemia and the number of ookinetes produced. The methods for in vitro ookinete formation and for estimating ookinete yields enabled us to study aspects of functional maturity of gametocytes independent of mosquitoes. The numbers of exflagellating gametocytes and in vitro ookinete yields in tail blood corresponded with those in heart blood and blood ingested by mosquitoes, suggesting a random distribution of functionally mature gametocytes within the vertebrate host.

Unisexual females, (80–100 days post-infection) were cultured in vitro and exposed to mature male worms from either mixed or single-sex infections. A comparison was made of the uptake of [3H]thymidine (used as a marker for DNA synthesis) by isolated unisexual females or by those females exposed to males derived from either mixed or unisexual infections during in vitro culture. All males were observed to be paired with females by approximately 18 h post-culture. At 24 h post-culture females paired with males from mixed infections had an elevated uptake of [3H]thymidine compared to females that had not paired and uptake was also significantly different (P ≥ 0–001) from females exposed to males from unisexual infections. By 48 h there was no significant difference between uptake in females paired with either type of male. The frequency of uptake in the paired females was approximately double that in unstimulated females while non-paired females in the presence of males had an increase of between 35 nd 45%. In a second experiment the effect of depriving a mature egg-laying female of the paired male was investigated. These females had a continual decrease in uptake of [3H]thymidine compared to mature females maintained with male partners. The uptake of [3H]thymidine was used as a marker for DNA synthesis and the effect of the male worm on DNA synthesis in the unisexual female is discussed in view of the above results.

Ookinetes have been cultured in vitro using modifications to the method of Weiss & Vanderberg (1977). Significant improvements in technique were produced by culture in medium at pH 8.4 and at a blood dilution at or over 1/10. Ookinetes produced were infective to mosquitoes by membrane feeding techniques. Ultrastructural analyses were made of nuclear, cytoskeletal, crystalloid and microneme development. The first intranuclear division in the zygote has been recognized as meiosis. Chromosome condensation during prophase follows the classical stages of leptotene, zygotene and pachytene. Diplotene and diakinesis are not present-the synaptonemal complexes persist into metaphase 1. Chromosomes separate at anaphase and rapidly de-condense prior to telophase. We have not recognized a second meiotic division in the ookinete. The implication of these findings to the molecular and Mendelian organization of the parasite genome are discussed.

This ultrastructural study has shown that there is a layer of dense flocculent material on the surface of juvenile Fasciola hepatica incubated in vitro with specific bovine antiserum. This material corresponds to the complexes of secreted glycocalyx and bovine antibody previously characterized by fluorescence microscopy. Bovine eosinophils attach closely to those regions of the parasite's surface that are free of flocculent precipitates. This close attachment is followed by degranulation of the eosinophils into the narrow zone between the cells and the parasite. Only in these regions is damage, in the form of vacuolation of the tegument, seen within the juvenile F. hepatica. It is concluded that the inability of bovine eosinophils to kill juvenile F. hepatica in the presence of specific antiserum results from the presence of a protective layer, consisting of antigen/antibody complexes, on the parasite's surface.

Medication of chicks with 70p.p.m. arprinoeid in the food, starting 2 days prior to inoculation with Eimeria tenella, resulted in decreased oocyst production and oocyst sporulation. The main morphological alteration seen during the development of the second-generation schizont was a reduction in the number of second-generation merozoites due to incomplete merogony, resulting in large masses of vacuolated residual cytoplasm. In places around the developing macrogamete the integrity of the parasitophorous vacuole was lost by either the host cell limiting membrane making contact with the parasite pellicle, or the disruption of one or both of these surfaces apparently brought about by the intervention of the host cell mitochondria. Thirty p.p.m. arprinocid reduced sporulation of the oocysts of E. brunetti and caused enlargement of the perinuclear space in the late merozoites and swelling of the endoplasmic reticulum around wall-forming bodies II.

Ten colostrum-deprived, coccidia-free lambs were reared artificially in indoor cages. At 4 weeks of age they were allocated to 5 groups of 2: Groups 1–3 received 104 105 and 106 sporulated oocysts of Eimeria erandallis/lamb respectively, each as a single inoculum. Group 4 lambs received daily doses increasing over a 4-week period. Groups 1–3 showed diarrhoea and weight loss from about day 14, followed by slow recovery from about day 30. The diarrhoea was accompanied by a wave of oocyst output and a reduction in serum albumin and plasma alkaline phosphatase activity. One lamb in group 4 died of an unrelated condition on day 15; the other showed no clinical effect of the oocyst inoculations and its oocyst output was related only to the first week or two of inoculation. Lambs in Group I showed no clinical effect or oocyst output following challenge with 106 oocysts on day 28. E. crandallis showed high pathogenicity and immunogenicity. Its pathogenicity was not closely related to infecting dose within the range 104–106.

Raillietiella gigliolii is a cephalobaenid pentastomid which inhabits the lungs of the South American worm-lizard Amphisbaena alba. The host is a facultative inquiline of nests of the leaf-cutting ant Atta cephalotes where it feeds occasionally (and possibly by accident) on ants but more often on beetles and their larvae which are themselves inquilines of ant nests. Ants store exhausted leaf-substrate in special underground chambers which serve as refuse dumps and it is here that larvae of the three-horned rhinoceros beetle Coetosis biloba feed: these larvae are also known to be prey items of A. alba. From observations of captive colonies of Atta, we have demonstrated that pentastomid-egg contaminated faeces of A. alba, introduced into the colony, are rapidly cut up and thrown onto the refuse dump, where, under natural circumstances they will be eaten by Coelosis. These larvae have an unusual and highly specialized gut physiology and parasite eggs will develop to an infective stage within the haemocoel in 70–96 days. Cockroaches are refractory to infection. Ants are the vital link in transmission since they literally deliver eggs to the Coelosis larvae. The strong trophic links which exist between the various components of the life-cycle offset a low fecundity of 100 eggs/female parasite/day but nonetheless maintain a high prevalence (86%) of infection.

The SICA[ − ] or non-agglutinable phenotype of Plasmodium knowlesi schizont-infected erythrocytes has been defined serologically but not biochemically. Similarly, non-cloned SICA[ + ] or agglutinable parasites have been shown serologically to express SICA or variant antigen(s) but the number and nature of such antigens have not been defined. Here we describe the immunochemical analysis of surface antigen expression on [125I]lactoperoxidase-labelled erythrocytes infected either with a SICA[ − ] elone or with non-cloned SICA[ + ] parasites using the methods developed for identification of variant antigens with cloned SICA[ + ] parasites. No 125I-labelled antigens in the size range Mr 190000–225000 were specifically immunoprecipitated from erythrocytes infected with the SICA[ − ] elone, even using homologous antisera produced by multiple infections or immunizations. Further, no125I-labelled proteins of this size were seen in detergent extracts of the SICA[ −] parasites that were not also seen with uninfected cells. We conclude that the SICA[ − ] phenotype reflects the absence of a variant antigen at the erythrocyte surface, as predicted by the serological assays. In contrast, with the non-cloned SICA[ + ] parasites, a complex group of proteins, Mr 195000–225000, was identified by [125I]lactoperoxidase labelling of intact infected erythrocytes. These proteins are SICA antigens since they not only share the characteristic detergent solubility properties and size range of SICA antigens identified previously with SICA[ + ] clones, but they were only immunoprecipitated by antisera which reacted specifically with the surface of infected erythrocytes. Agglutinating sera immunoprecipitated several of these 125I-labelled antigens. Sera specific for clones derived from this non-cloned SICA[ + ] population failed to agglutinate, but did react by indirect immunofluorescence with 10–16% of infected cells. These sera specifically immunoprecipitated single, quantitatively minor 125I-labelled antigens in this size range. The results suggest that a population of non-cloned SICA[+] parasites contains at least 10 different variant-antigen phenotypes. Indirect immunofluorescence was also performed against a non-cloned SICA[ + ] population derived by antigenic variation of a SICA[ + ] clone in vivo. The variant population contained at least 3 antigenically distinct SICA phenotypes, indicating that antigenic variation of clones may produce populations as antigenically heterogenous as antigenic variation of uncloned lines. It is therefore likely that natural malaria isolates contain a large number of different variant antigens.

Lung flukes, Paragonimus ohirai, from Kinosaki, Japan, characterized by 3 electrophoretic variants, FF, FS and SS, of glutamic-oxaloacetic transaminase (GOT: EC 2.6.1.1) were crossed in the laboratory. In the case of a cross between FS and SS, a parent, FS, produced 9 SS and 16 FS clones in the offspring, numbers not significantly different from that expected (1:1) from Mendelian inheritance. From the other cross FS × FF, 20 clones originating from the respective 20 miracidia were obtained from a parent FS. The phenotype segregation ratio was 6 FF and 14 FS, which is not significantly different from Mendelian inheritance expectation. Only 3 clones were obtained from the other parent FF, but these segregated in a ratio of 2 FS: 1 FF. These breeding data indicate that the GOT isozymes of P. ohirai are controlled by 2 codominant alleles, GotF and Gots at a single locus whose products aggregate randomly, forming a dimer.

Significant suppression in the incidence of cyclical development of Trypanosonia congolense, T. vivax and T. brucei occurred in Glossina morsitans centralis maintained on goats immunized with in vitro-propagated uncoated forms of T. congolense, T. vivax and T. brucei, respectively. This was observed when tsetse given a T. congolense-infected feed were subsequently maintained on uninfected immunized goats and also when uninfected tsetse were fed on immunized goats infected with T. congolense, T. vivax and T. brucei. Suppression of infection rates in tsetse was trypanosome species specific, but was independent of the trypanosome stock used for immunization of goats. These findings were reflected in antibody responses to uncoated trypanosomes, as measured by immunofluorescence and the solid-phase immuno radiometric binding assay. Thus, antibody from goats immunized with uncoated trypano somes of one species exhibited minimal reactivity with uncoated forms of other species of trypanosomes, but showed high levels of activity with uncoated forms of the same or unrelated stocks of the same species. However, in view of the range of hosts upon which tsetse feed, it is open to question whether the use of a vaccine which suppresses trypanosome infection rates in tsetse would have any significant effect in the field.

Plasmodium berghei XAT, an attenuated variant of lethal P. berghei, causes a resolving infection in Balb/c mice from which they recover in about 3 weeks. The parasitaemia displays an early peak at about 5 days, followed by a steep drop in parasite number associated with the appearance of degenerating forms inside mature erythrocytes; the parasites remaining are inside reticulocytes. By contrast, no degenerating parasites were seen in infections caused by the virulent parent, which was mainly confined to mature erythrocytes. However, P. berghei XAT was no more sensitive to reactive O2 metabolites, generated by alloxan, or to tumour necrosis serum, than its virulent parent. Furthermore, its early drop in parasitaemia was unaffected by silica. The drop still occurred in the absence of T cells, although the infection was then ultimately lethal, and it was not mediated by NK cells since it occurred in nude mice treated with anti-asialo GM1 serum to abolish NK cell activity. However, it was absent in splenectomized mice, in which P. berghei XAT infection was lethal. Thus, the attenuation of P. berghei XAT infection is not due to increased susceptibility to some of the agents thought to cause parasite destruction, but to some other mechanism in which the spleen is involved.

The effects of changes in red cell membrane properties on invasion by Plasmodium falciparum have been studied by varying the cholesterol content and the intracellular concentration of polyamines. Increased cholesterol content is known to cause large reductions in the internal fluidity of the phospholipid bilayer and a change in its preferred direction of bending, but does not cause changes in gross mechanical rigidity. Polyamines, on the other hand, are thought to increase the cohesion of the membrane cytoskeleton and impede translational diffusion of transmembrane particles, as well as increase trie mechanical rigidity of the membrane. Cells with membranes augmented by 50% in cholesterol show no reduction in their susceptibility to parasitic invasion, whereas an increase in cytosolic polyamine (especially spermine) concentration leads to strong inhibition of invasion. In neither case is the development of the intracellular parasite affected. We conclude that it is the macroscopic, rather than the microscopic rheoelastic properties of the membrane that influence the invasion process. Depletion of membrane cholesterol leads to a substantial reduction in parasitaemia; it is suggested that this is linked to the reduced phosphorus incorporation into spectrin in these cells. Polyamines may exert a significant effect at physiological concentrations and the possibility must be considered that the elevated polyamine levels found in red cells in sickle cell disease may account for the protection against P. falciparum.

The intraerythrocytic multiplication of two strains of Theileria annulata was studied with parasites maintained in stationary cultures and in the blood of infected cattle. In cultures established with blood from infected cattle 20–60% of single T. annulata piroplasms divided into quadruplet forms by day 6 in vitro. Transmission electron microscopic studies of T. annulata in culture showed that piroplasms possess intracytoplasmic food vacuoles and cytostomes during a pre-division trophozoite stage. The onset of intraerythrocytic multi plication was marked by the appearance of rhoptries and electron-dense plaques beneath the parasite's plasmalemmal membrane. The plaques developed into short segments of subplasmalemmal double membranes which were closely associated with the rhoptries. It was concluded that multiplication of T. annulata in erythrocytes occurred by schizogony, as nuclear division preceded cytoplasmic division and the final separation of merozoites. The four merozoites produced by intraerythrocytic schizogony had the same ultrastructural features as the T. annulata merozoites produced by intralymphocytic schizogony. Clusters of four merozoites, identical to those observed in stationary cultures, were also seen in the erythrocytes of persistently infected cattle and appeared to represent the most significant dividing forms of T. annulata in vivo.