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.

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.

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.

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.

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.

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.

Cercarial transmission of Schistosoma mansoni and S. mattheei was monitored in two small rivers near Durban, South Africa. The seasonal patterns recorded corresponded to those already documented for these parasites. Tn the case of S. mansoni, however, this was interrupted at the height of the transmission season. The reason for this was believed to be very low oxygen concentrations in the snail habitat due to unusually extensive growth of the plant Ludwigia stolonifera over the water. The failure of the spring rains, which would normally have flushed the system is seen as contributing to this phenomenon. Infection rates in the snail intermediate hosts were low ( > 10%). A preponderance of male worms of both schistosome species was noted.

The gross- and histopathology of natural and experimental Schistosoma curassoni infections in sheep were studied. The data obtained showed that S. curassoni infection in sheep causes only slight clinico-pathological manifestations with preferential involvement of the liver, the lower intestine and the urinary bladder. A variable spectrum of host reaction to the eggs within an individual animal was observed, reflecting the duration of presence of eggs in the organs. In the liver, egg granulomas were most numerous in the perilobular regions, while in the intestine, lesions were most pronounced in the mucosa of the rectum. The presence of eggs in 10% of the urinary bladders examined indicated some bladder involvement.

Mice of the inbred P strain fail to develop significant resistance to challenge Schistosoma mansoni infection at 6 weeks after either low-grade primary infection or vaccination with attenuated homologous parasites, in contrast to other strains such as C57B1/6, and thus provide a model for comparison of potential immune resistance mechanisms in low versus high responder animals. In this study, the antigen-specific cellular responses found to correlate with resistance in these strains were delayed cutaneous hypersensitivity, production of macrophage activating lymphokine and macrophage larvicidal activity, all of which were greater in infected or vaccinated C57B1/6 mice than in similarly immunized but non-resistant P mice. Humoral responses correlating with resistance were IgM reactivity to schistosomula surface antigens in both infected and vaccinated animals, as well as both IgM and IgG reactivity to soluble schistosome antigens in infected mice. Immune responses that showed no relationship with resistance included IgG reactivity to larval surface antigens and immediate hypersensitivity to soluble worm antigens. In infected mice, neither granuloma size nor extent of hepatic fibrosis correlated positively with resistance to challenge infection. Thus, similarities exist between patterns of resistance and immune response at this early time after immunization with either viable or attenuated parasites. These observations suggest that common immune effector mechanisms could be involved, with activated macrophages playing a central role in resistance.

A number of published studies of competition between parasite species are examined and compared. It is suggested that two general levels of interaction are discernible: these correspond to the two levels of competition recognized by workers studying free-living animals and plants: ‘exploitation’ and ‘interference’ competition. The former may be defined as the joint utilization of a host species by two or more parasite species, while the latter occurs when antagonistic mechanisms are utilized by one species either to reduce the survival or fecundity of a second species or to displace it from a preferred site of attachment. Data illustrating both levels of interaction are collated from a survey of the published literature and these suggest that interference competition invariably operates asymmetrically. The data are also used to estimate a number of population parameters which are important in determining the impact of competition at the population level. Theoretical models of host-parasite associations for both classes of competition are used to examine the expected patterns of population dynamics that will be exhibited by simple two-species communities of parasites that utilize the same host population. The analysis suggests that the most important factor allowing competing species of parasites to coexist is the statistical distribution of the parasites within the host population. A joint stable equilibrium should be possible if both species are aggregated in their distribution. The size of the parasite burdens at equilibrium is then determined by other life-history parameters such as pathogenicity, rates of resource utilization and antagonistic ability. Comparison of these theoretical expectations with a variety of sets of empirical data forms the basis for a discussion about the importance of competition in natural parasite populations. The models are used to assess quantitatively the potential for using competing parasite species as biological control agents for pathogens of economic or medical importance. The most important criterion for identifying a successful control agent is an ability to infect a high proportion of the host population. If such a parasite species also exhibits an intermediate level of pathology or an efficient ability to utilize shared common resources, antagonistic interactions between the parasite species contribute only secondarily to the success of the control. Competition in parasites is compared with competition in free-living animals and plants. The comparison suggests further experimental tests which may help to assess the importance of competition in determining the structure of more complex parasite-host communities.

The immune status of a group of sheep naturally infected with hydatid cysts was investigated. Low specific anti-hydatid antibody (Sab) titres and negative results in intradermal and leucocyte migration tests occurred in hosts with high hydatid antigen-stimulated lymphocyte transformation(LT). Total(non-specific) IgG was elevated in infected sheep and was positively correlated with LT. Circulating hydatid antigen (cAg) was detectable in some infected sheep, but not in all of those with low Sab. Relationships between eAg, Sab and immune complex levels suggest that most eAg is present in immune complexes.

During the course of a primary infection of Nippostrongylus brasiliensis in protein-malnourished rats, plasma concentrations of corticosterone were found to decrease from 0.89±0.03 at the start of the infection to 0.28±0.08μmol/l, 9 days post-infection (p.i.). Similarly, adreno-corticotrophic hormone levels were also observed to fall from 62.09±12.06 to 20.54±1.81 by 5 days p.i. and to 35.12±16.61 nmol/l by 9 days p.i. However, no significant changes were seen in the dry weight of the adrenal glands other than those expected in relation to changes in body weight. Concentrations of both plasma tri-iodothyronine and thyroxine were also severely depleted over the same period from 5.33±0.34 to 1.09±0.42 and 104.43±10.4 to 34.12±8.95 nmol/l respectively. This was considered not to be due to any change in the capacity of the plasma to bind these hormones. Results for insulin were highly variable, but overall were lower from protein-malnourished rats than results documented for well-nourished rats and a reduction from 18.07±3.17 to 6.08±2.18μunits/ml, 9 days p.i. was observed. The results are discussed in relation to the role of these hormones in protein metabolism in a protein-malnourished animal.

Examination of the short-term interaction of the haemocytes and lysozyme of Galleria mellonella larvae with the entomogenous nematode Steinernema feltiae DD136, in vitro revealed that the nematodes did not reduce the adhesion of Bacillus subtilis or Xenorhabdus nematophilus subsp. nematophilus to larval granulocytes or plasmatocytes. There was no evidence of humoral, sheath or cellular encapsulation of S. feltiae in the haemolymph in vitro or in vivo. Compared with the phosphate-buffered saline-injected larvae the axenic nematodes did not alter the total or differential haemocyte counts during the initial 4 h of parasitism. The ability of the insect larvae to remove B. subtilis and X. nematophilus from the haemolymph was not influenced by axenic S. feltiae. The bacteria from the intestine of surface disinfected, monoxenically cultured S. feltiae elevated the larval total haemocyte counts and damaged the haemocytes. The activity of larval lysozyme was not influenced by axenic S. feltiae.

The influence of a chronic subclinicael infection of Trichostrongylus colubriformis, 2500 larvae/day for 12 weeks, on gastrointestinal motility and digesta flow was studied in 12 sheep supplied ad libitum with food and water. Motility was recorded by X-radiography and electromyography from chronically implanted electrodes;abomasal volume and outflow were estimated by dilution of CrEDTA; small intestinal transit time was estimated by passage of Phenol Red. The findings were compared with measurements made prior to infection at restricted food intake and reported separately. The first effects of infection were seen after 3–4 weeks. No animal developed diarrhoea, but food intake was progressively reduced. Small intestinal transit time, abomasal volume and half-time of marker dilution increased while abomasal outflow decreased during infection. These changes occurred both in absolute terms and when compared with values predicted from the observed level of food intake. As the animals became resistant to the parasites abomasal volume and digesta flow returned towards control values (weeks 10–12). The migrating myoelectric complex (MMC) was disrupted in only one sheep, and only transiently. In all sheep the frequency of the MMC was increased during infection and there was a progressive inhibition of abomasal, duodenal and jejunal motility. X-radiography showed there was prolonged pooling of digesta in the proximal small intestine which was cleared only at the phase of regular spiking activity. Two sheep given an anthelmintic drench recovered normal motility and clearance of digesta. It is concluded that subclinical infection of sheep with T. colubriformis alters the normal pattern of gastrointestinal motility in the absence of any diarrhoea, and causes inhibition of abomasal and proximal small intestinal motility and digesta flow. The increased frequency of MMCs helps to maintain digesta flow through the proximal small intestine.

The fully embryonated egg of Notocotylus attenuatus is shown to contain a sporocyst, an opercular cord and apparently 2 cells which are situated outside the sporocyst. One of these cells is rich in glycogen whereas the other contains a variety of inclusions. These contents are indicative of a truncated life-cycle which omits the miracidium, an observation as yet unique among the Digenea.