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.

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.

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.

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named ‘megasomes’. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

The localization of host (mouse)-related DNA sequences in the adults and cercariae of Schistosoma japonicum and S. mansoni was examined by in situ hybridization using 32P-labelled probes. The hybridization signals to the sequences of the mouse type C and type A retroviruses were clearly observed in the subtegumental layer and inner tissues of S. japonicum adults. In contrast, it was hard to find any signals to these sequences in S. mansoni adults. Distinct signals to the env-specific region of the mouse ecotropic type C retrovirus were observed in the subtegumental layer and inner tissues of S. mansoni adults. No signal to the sequence of the mouse type B retrovirus was found on the sections of either schistosome adult. The signals to the sequence of the genomic clone SmM51 derived from male S. mansoni were found in the tissues of both the schistosome adults. The signals to the sequences of the mouse type A and env-specific region of ecotropic type C retroviruses were not seen in the cercariae of each species, whereas the signals to the sequence of SmM51 were detected in S. mansoni cercariae. These observations suggest that host-related DNA sequences might be incorporated unequally into the schistosome genome during development in the final host.

The entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

For the description of the dynamics of snail infection by the 1st-stage larvae of protostrongylid nematodes, Skorping (1988) used the miracidia-snail model (Anderson, 1978). Here it is shown that, in contrast to miracidia, in protostrongylids the instantaneous rate of infection, α, is strongly dependent on the experimental design (factors like host size and size of the experimental arena). With respect to this, Anderson's model is modified by incorporation of the experimental design. The parameter α in its new sense as the rate of penetration (probability that the infective larva will penetrate into the host during a time unit) is shown to remain dependent, although much less so, on the experimental design. Only the inclusion of the assumed effect of mucus, which decreases the rate of penetration, yields a parameter α0 (the initial rate of penetration), which is completely independent of the design of the experiment, is species-specific, and also gives the best fit to the empirical data. As the above-mentioned factors can strongly influence the value of the instantaneous rate of infection in the laboratory experiments, α0 is more suitable as a measure of either the larval infectivity for the snail or snail susceptibility to infection by the protostrongylid larvae.

The surface of the cuticle of adult Nippostrongylus brasiliensis has been studied by means of the freeze-fracture technique and by transmission electron microscopy. Some of the surface coat appears to have been shed from the surface of the cuticle of adults fixed in situ in the intestine of its host and from the surface of individuals removed from the intestine and freeze-fractured. Freeze-fracturing the cuticle of individuals removed from the host has shown that this surface coat varies in thickness from 30 to 90 nm. The epicuticle is about 20 nm thick and cleaves readily to expose E- and P-faces. The P-face of the epicuticle possesses a small number of particles, similar to intra-membranous particles, whilst the E-face possesses a few, widely scattered depressions. Despite the presence of these particles the epicuticle is not considered to be a true membrane. Freeze-fracturing the remainder of the cuticle has confirmed its structure as described by conventional transmission electron microscopy. Clusters of particles on the P-face of the outer epidermal (hypodermal) membrane and corresponding depressions on the E-face of the membrane are thought to be associated with points of attachment of the cuticle to the epidermis (hypodermis). No differences in appearance of the cuticle and its surface layers were observed in individuals taken from 7-, 10-, 13- and 15-day infections.

The amounts of acetylcholinesterase (AChE) in homogenates of infective-stage larvae of strains, from different countries, of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis resistant or susceptible to benzi-midazole (BZ) anthelmintics, were compared using a rapid colorimetric assay. The levels of AChE were much greater in those strains which were BZ-resistant than in BZ-susceptible populations. The larvae of susceptible and resistant H. contortus contained less AChE than did the larvae of the other two species. One-way analysis of variance showed that the observed differences between susceptible and resistant strains were significant in each of the species studied. It is suggested that the extra enzyme may act as an anthelmintic-detoxifying enzyme, may inactivate the active site of the anthelmintic or may prevent binding of the anthelmintic to its receptor

Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions. In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved. This showed that external alkaline pH and high sodium ion concentration were highly stimulatory. The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells. The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory. Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.

Random amplified polymorphic DNA (RAPD) offers a potential basis for the development of a diagnostic assay to differentiate the potato cyst nematode species Globodera rostochiensis and G. pallida. Nine decamer primers have been tested for their ability to amplify species-specific DNA sequences. Primer OPG-05 produced 2 discrete DNA fragments, which were consistently present in 5 G. rostochiensis populations and absent in 5 G. pallida populations. These fragments were detectable in single females as well as in single 2nd-stage juveniles. Their amplification is extremely efficient, and reproducible over a wide range of template concentrations. One-fifth of a single juvenile is sufficient to generate reproducible RAPD markers. The amplification from single juveniles requires no DNA isolation. The use of a crude homogenate does not impair the polymerase chain reaction.

Indirect immunofluorescent studies have been used to localize a glycoprotein (gp32) in the amphids of 2nd-stage juveniles (J2) of Meloidogyne incognita parasitic in the roots of tomato plants, male Meloidogyne javanica and some young adult female M. incognita. Immuno-electron microscopic studies showed an apparent absence of gp32 in the amphids of mature adult female M. incognita. Chemoattraction experiments demonstrated that the binding of gp32 antibodies to the amphidial secretions of infective J2 M. incognita partly impaired the ability of the nematodes to locate tomato roots.