Prototheca richardsi, an unpigmented heterotrophic alga, causes growth inhibition in amphibian larvae and has proved refractory to culture in Vitro. P. richardsi replication is dependent on regular passaging through tadpole digestive systems; uptake of thymidine by free-living Prototheca cells and incorporation into DNA are very low by comparison with leucine uptake and incorporation into protein, but DNA synthesis is detectable in cells isolated from tadpole intestines. DNA replication was elicited 6–8 h after ingestion in protothecans fed to tadpoles and subsequently re-isolated from them, providing that the tadpoles were fed subsequent to the ingestion. It appears that passaging through tadpole intestines provides an essential stimulus to maintaining an active cell division cycle in P. richardsi.

A microsporidium of the genus Encephalitozoon was isolated into culture from the nasal epithelium of a patient with AIDS. It was compared with in vitro isolates of Encephalitozoon cuniculi and the type isolate of Encephalitozoon hellem by SDS–PAGE and by Western blotting with murine antisera raised to E. cuniculi, E. hellem and the nasal isolate, monoclonal antibodies raised to E. cuniculi and sequential sera from the patient. All tests showed similarities between E. hellem and the nasal isolate but differences between these two isolates and E. cuniculi. Minor protein differences between E. hellem and the nasal isolate were not considered sufficient to separate them at the specific level. The new isolate is named the Wainwright isolate of E. hellem. The ultrastructure of the Wainwright isolate in vitro was similar to that of the parasite in vivo but there was a greater tendency for disruption of the parasitophorous vacuoles. The deposition of the electrondense surface coat on the sporogonic stages of E. hellem, as a uniform layer which later thickens, is in contrast to its deposition as broad bands, which later join up, in E. cuniculi. This may be a useful character in distinguishing the species without recourse to analysis of protein profiles.

This study is concerned with the influence of testosterone on the phospholipid class and the phosphatidylcholine molecular species composition of various fractions obtained from the blood of Plasmodium chabaudi-infected mice. Blood plasma, infected erythrocytes, isolated parasites and erythrocyte membranes isolated from both non-infected and infected erythrocytes in the form of ghosts were analysed. In general, the phospholipid classes remained unaffected, while the phosphatidylcholine (PC) molecular species composition showed differences after testosterone treatment. In infected erythrocytes, there was a decrease in 16:0/20:4-PC and 18:0/20:4-PC and an increase in 16:0/18:2(16:0/20:3)-PC. The decrease of 16:0/20:4-PC was exclusively confined to parasites. The rise in 16:0/18:2(16:0/20: 3)-PC and the diminution of 18:0/20:4-PC occurred in the erythrocyte membrane of both infected ghosts and non-infected ghosts as well as in the blood plasma. It is suggested that these changes occur primarily in the plasma thereby influencing the erythrocyte membranes. The decrease in 16:0/20:4-PC supports the view of the independence of the parasite from the biosynthetic lipid pathways of its host cell.

The mini-exon gene repeats from three different strains of Endotrypanum schaudinni have been amplified by the polymerase chain reaction (PCR). Sequence analysis of the cloned products shows the gene and intergenic region to be identical in two of the strains (LV86 and M6159); the intergenic region from the mini-exon gene of the third strain (LV59) is significantly different. The LV86 gene and an intergenic probe from the LV59 mini-exon gene do not cross-hybridize with the mini-exon gene from New World Leishmania species. These data provide the basis of a PCR assay to detect E. schaudinni and distinguish it from New World Leishmania species, and which should be applicable to epidemiological studies in insect vectors and mammalian reservoirs. The identification of two different mini-exon gene repeats in E. schaudinni isolates is indicative of further strain variation within this species.

Plasmodium falciparum, P. knowlesi and P. chabaudi showed a significant activity of methylenetetrahydrofolate reductase (MTHFR). The presence of this enzyme completes the methionine synthesis cycle, in which the one-carbon fragment from serine side-chain can be transferred to methionine. However, while metabolic labelling of methionine from l-3 [14C]serine could not be demonstrated in P. falciparum, the significance of MTHFR was implicated by a novel pathway for salvage of exogenous 5-methyltetrahydrofolate from the host cell. The methyl group of the cofactor was incorporated into methionine, and the folate cofactor was found in the same pool as that derived from de novo synthesis with paminobenzoic acid as the precursor, shown previously as polyglutamylated 5-methyltetrahydrofolate. It is proposed from these results that the function of MTHFR and the methionine synthesis cycle is not the supply of methionine, but the generation of active folate cofactors from more stable precursors salvaged by the parasites

Adhesion of parasitized red blood cells to vascular endothelium is thought to play an important role in the development of the ischaemic complications associated with severe falciparum malaria. Using a novel, flow-based assay, we have investigated the adhesion of parasitized red blood cells to formalin-fixed human umbilical vein endothelial cells (HUVEC), for isolates obtained from 32 Gambian subjects with mild or severe falciparum malaria. Red cells infected with wild strains of Plasmodium falciparum were able to adhere to HUVEC under physiologically relevant flow conditions, but the level of adhesion was highly variable, ranging from 1 to 688 adherent cells per mm2 of HUVEC. Within isolates, some adherent parasitized cells remained stationary, whilst other formed less stable interactions and rolled slowly over the cell surface. There was no significant difference in adhesion of parasitized cells between isolates obtained from mild or severe cases of malaria, although a subset of isolates did show very high levels of adhesion. The results suggest that there is not a simple relationship between the adhesion of parasitized cells to cultured endothelial cells (presumably via the receptor ICAM-1) and the clinical severity of the disease, although variation in microvascular adhesion in vivo may still be a determinant of ischaemic complications.

The effect of tumour necrosis factor-α on malaria-infected mice was studied. C57B1/6J mice infected with Plasmodium berghei K173 exhibited an increased sensitivity to exogenous TNF. Injection of 15 μg TNF was lethal to some of the animals when given 5–7 days after infection, while when given later on in the infection (i.e. days 8–10) amounts as low as 2·5 μg TNF appeared to be lethal in all mice. The pathology in infected mice treated with TNF resembled that found in the brains of infected mice dying with cerebral malaria. Infected mice treated with TNF, however, also developed severe pathological changes in other organs. On the contrary, treatment with sublethal amounts of TNF (1·0 μg or less) given on days 8 and 9 after infection, protected mice against the development of cerebral malaria. In addition, infected mice exhibited an enhanced sensitivity for treatment with lipopolysaccharide (LPS). Sublethal amounts of LPS, however, did not prevent mortality as in TNF-treated mice (LPS-treated mice died at about the same time as infected mice that developed cerebral malaria), but no cerebral haemorrhages were found in the majority of LPS treated, infected animals. Treatment with dexamethasone during infection protected mice against the development of cerebral malaria, but did not suppress their increased sensitivity to exogenous TNF. Treatment of mice with liposome-encapsulated dichloromethylene diphosphonate (lip-Cl2MDP), used to eliminate macrophages (an important source of TNF), prevented the development of cerebral malaria, but only when given before day 5 of infection. Mice protected by treatment with lip Cl2MDP, however, remained sensitive for LPS on the eighth day of infection.

Two clones of the human malaria parasite Plasmodium falciparum, denoted 3D7 and HB3, were grown in vitro under conditions permitting the development of gametocytes. The two clones differ in their allelic forms of two antigen genes MSP1 and MSP2. The alleles can be distinguished as size differences of polymerase chain reaction (PCR) amplified fragments of repetitive regions of each gene. Mosquitoes (Anopheles stephensi) were fed on a mixture of these gametocytes. A total of 128 oocysts was isolated from the midguts of infected mosquitoes from 9 crossing experiments between the clones. DNA extracted from these oocysts was amplified by PCR. Oocysts which contained both alleles of each gene (MSP1 and MSP2) had developed from heterozygotes produced by cross-fertilization events between 3D7 and HB3 gametes. The remaining oocysts contained single alleles of each gene, in parent clone combinations, and these had developed from homozygotes formed by self-fertilizations. The results suggest that gametes in the original mixture fed to mosquitoes had undergone random mating.

The effects of purified monoclonal immunoglobulins from control, or transmission-blocking anti-Pbs21 antibodies, upon the infection of Anopheles stephensi by ookinetes of Plasmodium berghei are compared. Anti-Phs21 antibody reduced mean intensity and prevalence of infection by 94·7 and 58·7% respectively if added to the infectious bloodfeed at a concentration of 100 μg/ml. Fab fragments were of similar efficacy. No transmission enhancement was detected with declining antibody concentrations. Addition to subsequent (second) feeds reduced mean oocyst intensity but not prevalence. The reduction in blockade declined from 41% at day 2, to 4% at day 8. Second bloodfeeds, containing control globulin taken 4 or 6 days (but not 2 days) after infection, increased sporozoite burden in the salivary glands. At all times anti-Pbs21 reduced sporozoite number in the thorax compared to time-matched controls, but again highest gland intensities were obtained when the second bloodfeed was given on day 4. We conclude that second bloodfeeds containing transmission-blocking antibody simultaneously serve two opposing roles, (1) inhibition of parasite development and (2) the supply of nutrients which permit more sporozoites to be produced by each oocyst.

CBA/T6 strain mice infected with Plasmodium berghei ANKA develop cerebral symptoms and die, with mononuclear cell attachment to the cerebral microvascular endothelium, petechial haemorrhages and breakdown of the blood–brain barrier, some 6–7 days post-inoculation. The effects of dietary restriction on this process were examined. Mice were fed ab libitum (Group 1) or their food was restricted to produce body weight loss of 1·0–2·0 % (Group 2), 2·5–3·5 % (Group 3), 4·0–6·5 % (Group 4) or 7·0–9·5 % (Group 5) relative to Group 1. Dietary restriction reduced deaths caused by cerebral malaria from 100% in Group 1 to 47% (Group 2), 43% (Group 3), 10% (Group 4) and 53% (Group 5). Restriction of food intake had no effect on (1) the progression of parasitaemia in infected mice (2) changes in haematocrit, spleen weight, total lymph node cell number or (3) peritoneal exudate cell number in either malaria-infected or uninfected mice. P. berghei ANKA infection did not significantly affect the proportion of lymph node leucocytes that were Thy-1+T cells or CD8+T cells, but did lead to significant increases in the CD4+ and B cell populations. Dietary restriction alone increased the lymph node CD4+cell population but did not affect the increase in B cells in malaria-infected mice. P. berghei ANKA infection and dietary restriction together did not lead to increased CD4+cell numbers in lymph node leucocytes. The in vitro proliferative response of isolated lymph node cells to concanavalin A or phorbol myristate acetate plus ionomycin was measured and found to be identical in all treatment groups. Plasma levels of tumour necrosis factor (TNF) increased from undetectable in uninfected mice or P. berghei ANKA-infected mice on days 1–6 post-inoculation to 21 ± 2 ng/ml on day 7 when cerebral symptoms were at their height and death was imminent. This increase in plasma TNF was substantially inhibited in those mice subjected to regimes of dietary restriction. There was a good correlation (r2 = 0·73) between plasma TNF level and percentage mortality in the 5 treatment groups. The results indicate that dietary status is an important factor in the outcome of murine cerebral malaria and perhaps, by extrapolation, in the human disease.

CBA/T6 mice inoculated with Plasmodium berghei ANKA strain (PbA) exhibited cerebral symptoms and died from cerebral malaria 6–8 days p.i. whereas DBA/2J mice developed (around days 6–9) a non-fatal cerebral malaria, with milder cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop cerebral malaria. These mouse/parasite strain combinations were used, in conjunction with the retinal whole-mount technique, to elucidate factors critical in the pathology of murine cerebral malaria. CBA/T6 mice infected with PbA (PbA-CBA mice) demonstrated mild changes in vascular permeability as early as days 2–3, prior to the appearance on day 5 of cerebral symptoms, whereas mice with non-cerebral malaria did not show any vascular permeability changes until the very late stage of the disease (days 14–22). In the PbA infections, progressive deterioration of endothelial barrier properties, demonstrated by Evans' Blue leakage both generally and from specific focal areas, as well as a developing monocytosis and adherence of mononuclear cells to the endothelium of the retinal vessels continued until death (in CBA/T6 mice) or resolution (in DBA/2J mice). Adherent monocytes, particularly in PbA-CBA mice, were associated with reduced Hoechst staining of individual endothelial cells and a banking up proximally of both parasitized and non-parasitized blood cells in the small blood vessels, often with accompanying focal leakage of Evans' Blue from the retinal vessels. The occurrence and severity of these early changes in the microcirculation correlated with the subsequent development of cerebral symptoms. Monocyte margination appeared to be the most significant factor associated with the development of cerebral symptoms.

A DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

The gene for the 32 kDa surface protein (p32) of Theileria sergenti was cloned into λgt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected the Pst I-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and that transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

A total of 21 Theileria parva stocks from 6 countries were characterized using T. parva repetitive and ribosomal DNA probes, a Plasmodium berghei telomeric oligonucleotide and a panel of anti-schizont monoclonal antibodies (MAbs). Hybridization of the repetitive DNA probe to Southern blots of EcoRI-digested T. parva DNA revealed 20 different restriction fragment patterns among DNA samples isolated from infections initiated using 16 parasite stocks. The panel of anti-schizont MAbs defined 8 different profiles among schizont-infected lymphoblastoid cell-cultures infected with the same 16 T. parva stocks. Many stocks, which were differentiated by the repetitive DNA probe, could not be distinguished using the anti-schizont MAbs. A cloned T. parva small subunit ribosomal RNA (SSUrRNA) gene probe separated 17 T. parva stocks into 2 groups, exhibiting either 1 or 2 restriction fragments, when hybridized to EcoRI-digested T. parva DNA. When hybridized to PvuII-digested DNA from 8 T. parva stocks, the ribosomal probe identified 4 groups with similar restriction fragment patterns. A synthetic oligonucleotide derived from a P. berghei telomeric sequence hybridized to 7 or 8 size-polymorphic restriction fragments in the EcoRI-digested DNA of most T. parva stocks. The telomeric and ribosomal probes defined the same 4 groups among 8 T. parva stocks as assessed by similarities in restriction fragment patterns. Based on the comparison of repetitive DNA sequences from the T. parva Uganda and Muguga stocks, a synthetic oligonucleotide was developed which distinguished the DNA of the T. parva Uganda stock from that of 4 other T. parva stocks on a positive/negative basis.

A new isolate of Trypanosoma musculi has been derived from organisms recovered from Mus spretus captured in Lisbon, Portugal. The time-course and profiles of infection with this isolate in inbred mice have been compared with those obtained with the existing Partinico II isolate. Infections with the Portuguese isolate are less intense, and controlled more quickly than those with the Partinico isolate. As with the latter, there are marked mouse strain-dependent influences on infection with the Portuguese isolate, but these strain-dependent characteristics differ considerably with each isolate. For example, NIH mice were the most susceptible to the Partinico II isolate, but virtually refractory to the Portuguese isolate. Mice exposed to infection with one isolate show complete immunity to both homologous and heterologous challenge infections. These striking interactions between host and parasite genotype are discussed in terms of immunological influences on infection.

Athymic mice (BALB/c nu/nu/Ola/Hsd) were experimentally infected intraperitoneally with Nosema corneum spores. Infection was monitored in the first and second weeks post-infection. The liver, spleen, kidney, intestine, lung, heart, brain and eye were collected. Quantification of infection in each organ using three different techniques gave approximately the same pattern of infection. Infection increased with time. Histological observations were made on the sites of infection in each organ. All organs were infected, the liver being the most heavily infected. The eye was infected in the retina in contrast to the cornea which was the site of infection in the original host. The present study of N. corneum in athymic mice has shown that this system could also be used to study host-parasite relationships and serve as a model for testing therapeutic agents. Previously the only microsporidian serving as a suitable model for human microsporidiosis was Encephalitozoon cuniculi.

Nucleosome filaments of two stages of the life-cycle of Trypanosoma brucei brucei, namely bloodstream forms and procyclic culture forms, were investigated by electron microscopy. Chromatin of bloodstream forms showed a salt-dependent condensation. The level of condensation was higher than that shown by chromatin from procyclic culture forms, but 30 nm fibres as formed in rat liver chromatin preparations were not found. Analysis of histones provided new evidence for the existence of H1-like proteins, which comigrated in the region of the core histones in SDS–PAGE and in front of the core histones in Triton acid urea gels. Differences were found between the H1-like proteins of the two trypanosome stages as well as between the core histones in their amount, number of bands and banding pattern. It can be concluded that T. b. brucei contains a full set of histones, including H1-like proteins, and that the poor condensation of its chromatin is not due to the absence of H1, but most probably due to histone–DNA interaction being weak. It is obvious that structural and functional differences of the chromatin exist not only between T. b. brucei and higher eukaryotes, but also between various stages of the life-cycle of the parasite. It is therefore not adequate to investigate the chromatin only of the procyclic culture forms as a model for all stages of the life-cycle of T. b. brucei.