Recent data indicate that platelets may play an important role in the host defence against Toxoplasma gondii infections. T. gondii-stimulated human platelets release thromboxane A2 (TXA2) and 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid and 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid (Yong et al. 1991; Henderson et al. 1992). We have previously demonstrated that the eicosanoid TXA2 has potent cytotoxic activity against T. gondii trophozoites (Yong et al. 1991). In this study, we examined whether 12-HETE, 13-HODE, and linoleic acid also have toxoplasmacidal activity. 13-HODE at concentrations ≥ 10−8 M rapidly induced cytotoxic changes in T. gondii. Ultrastructural changes induced by 13-HODE in T. gondii included an initial leakage of cytoplasmic contents into a space between the inner and outer parasite bilayer membrane units which was followed by intracellular vacuolation and loss of cytoplasmic contents. In contrast, linoleic acid and 12-HETE lacked toxoplasmacidal activity at 10−10–10−6 M concentrations. These data indicate that 13-HODE, a product of linoleic acid metabolism, has potent cytotoxic activity against T. gondii; this toxoplasmacidal activity may be important in the inflammatory response to this pathogen.

The effect of treatment with monoclonal antibodies (Mabs) which deplete CD4+ or CD8+ T lymphocytes, on infections with Eimeria spp. was examined in NIH mice. Treatment with anti-CD4 Mab increased susceptibility to primary infections with E. vermiformis or E. pragensis and reduced the subsequent resistance of the mice to homologous challenge. Similar treatment of immune mice did not affect their resistance to re-infection but this was reduced in mice depleted of CD8+ T lymphocytes. In mice immunized with E. vermiformis the effect of CD8+-depletion was very slight, apparent only as the presence of small numbers of oocysts in the faeces of some mice; in mice immunized with E. pragensis there was a small, though significant, increase in oocyst production, compared with controls and anti-CD4-treated groups. These results confirm the importance of mechanisms involving the function of CD4+ T lymphocytes in the control of primary infections with Eimeria spp. and indicate that CD8+ cells play some part in the expression of resistance to reinfection. They also show that a major part of this resistance was not affected by either of the treatments given.

Erythrocyte invasion assays are described for two species of rodent malaria, namely Plasmodium berghei and P. c. chabaudi. These invasion assays are simple, are carried out using a candle jar and allow a number of assays to be performed simultaneously. Our results demonstrate that both rodent malaria species show an in vitro preference for reticulocytes although the preference of P. c. chabaudi for these cells is not as marked as that of P. berghei. The details of our invasion assays and our results obtained are discussed.

Cloned lines of the four rodent Plasmodium species can be differentiated by the RFLP pattern generated following Southern blotting and probing with PCsv4.1, a probe derived from a P. chabaudi chabaudi genomic library. Groups of CBA/Ca mice were inoculated simultaneously with cloned lines from two parasite species or strains. Six mixed species and three mixed strain infections using rodent malaria lines were initiated. The composition of the parasite population in each group was determined qualitatively and semi-quantitatively by analysis of the DNA purified from daily blood samples, thereby providing a dynamic representation of each mixed infection. Effects on the course of parasitaemias are presented and discussed.

This study examined whether allelic changes observed when clinical isolates of Giardia intestinalis made in suckling mice were adapted to in vitro growth occurred as a result of gene switching (alternate isoenzymes) or through selection of organisms with different genotypes from mixed infections. Samples were compared electrophoretically at 20 enzyme loci. Marked allelic differences were detected between the uncloned clinical isolates grown in mice and the axenic cultures established from them. Furthermore, the allelic profiles of the uncloned isolates changed during the course of in vivo or in vitro growth. In contrast, all clones produced from each isolate retained identical allelic profiles, regardless of whether they were grown in vivo or in vitro. These findings argue against gene switching as an explanation for the observed allozyme changes and support preferential selection of organisms with specific genotypes by growth conditions. The data indicate the presence of at least 2 and possibly up to 4 distinct genotypes within each clinical isolate. The genetic differences detected between clinical isolates in suckling mice were of similar magnitude to those that separate different axenic isolates of G. intestinalis into cryptic species. Conversely, the genetic differences between the isolates were limited when sampled after establishment in vitro. These findings have significant implications for research on Giardia and other medically important parasites and raise the possibility that culture may exert a similar selective bias on the genotypes isolated from infections with other parasitic protozoa.

Immunoprecipitation combined with electrophoresis in gelatin-polyacrylamide gels was successfully used for detection of antibodies against numerous proteinases of Trichomonas vaginalis in infected patients. The method proved to be highly specific as anti-proteinase antibodies were absent in women with negative cultivation of T. vaginalis and no history of trichomoniasis. Sera of 71% and vaginal washes of 86% patients with trichomoniasis were positive for these antibodies. In vaginal washes, but not in sera, antibodies were partly complexed with proteinases, possibly of trichomonad origin. It was also shown that serum antibodies as well as local anti-proteinase antibodies persisted for weeks after patients had been cured.

Cells of Leishmania donovani in co-culture with Trypanosoma brucei, were severely affected in their growth, resulting in swelling and subsequent lysis. Similar effects were also observed when Crithidia luciliae or Phytomonas sp. were co-cultured with T. brucei. Direct contact between the cells under investigation and T. brucei was necessary because T. brucei did not hamper the growth of the other trypanosomatids, when separated by a filter with 0.2 μm pore size. Examination of this phenomenon at the ultrastructural level, in a co-culture of L. donovani and T. brucei, suggests that the plasma membrane permeability is increased in the former, as a result of a close cellular contact between the two cell types.

The complete sequence of the cDNA encoding a 20 kDa calcium-binding protein of Schistosoma mansoni (Sm20) has been determined. The predicted amino acid sequence contains 4 EF hand domains but examination of the predicted secondary structure of Sm20, together with the specific residues in each calcium-binding domain, suggests that only 1 EF hand (domain IV) is functional. Sm20 is most homologous to calmodulin, troponin C and the regulatory light-chain of myosin, particularly those of invertebrates. However, troponin C and the regulatory light-chain of myosin can be distinguished from Sm20 by size and by their differential levels of expression during the life-cycle. Sm20 also appears to be distinct from calmodulin but may be functionally equivalent to the soluble sarcoplasmic calcium-binding proteins of molluscs and crustacea which may act as a reservoir for calcium in muscle. Sm20 is encoded by a small multi-gene family whose members are clustered within a 15 kb region of the genome. A 20 kDa antigen, cross-reactive with Sm20, is expressed in Schistosoma haematobium, Fasciola hepatica and Paragonomus mexicanus.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5–72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS–PAGE. The infected cells produced a number of extra activities at pH 8·0 in the presence of Ca2+. Calcium was found to enhance the activites but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

The humoral responses of Sprague–Dawley rats infected with Brugia pahangi were examined for up to 6 months after infection by ELISA, immunoblotting, and IFAT. In 2 experiments, 50% and 62·5% of rats developed patent, microfilaraemic infections. Mean adult worm burdens at autopsy were approximately 2% of the inoculum, and only patent rats yielded living adult worms. IgG antibody levels against crude somatic extracts (CSE) of all parasite stages and against adult excreted/secreted (ES) products were significantly higher in patent than non-patent rats. Both patent and non-patent rats produced anti-microfilarial surface antibody, as revealed by immunofluorescence. Immunostaining of Western blots by early infection sera showed no consistent difference in recognition of infective larval (L3) antigenic components by IgG or IgM antibody between eventually-patent and eventually-non-patent rats. By 26 weeks, however, patent rats recognized more components. The data suggest that antibodies against L3, adult, and microfilarial somatic antigens, ES antigens and microfilarial surface antigens do not correlate with the subsequent development of microfilaraemia in any individual rat.

The miracidium of Sanguinicola inermis contains a stylet and rodlet complex located within the single apical gland, which lies between a pair of lateral glands. The miracidial stylet consists of an arrangement of hollow tubes, composed of microtubules, connected at their base to large secretory granules and opening anteriorly through the apical papilla. A single rodlet lies adjacent to the stylet base and at least 8 rodlets are arranged at the apical tip of the stylet. The surface plasma membrane of the apical papilla is drawn into knob-like projections and 2 ciliated sensory endings are associated with the papilla. The ultrastructure of the apical gland, especially the stylet and rodlet complex, is related to the requirement for sanguinicolid miracidia to penetrate the double barrier of fish and snail hosts.

The survival, growth and development of Onchocerca lienalis 3rd-stage (L3) larvae implanted into mice within micropore chambers has been studied with a view to developing a vaccination model for studies of protective immunity in onchocerciasis. The influence of host genetics on worm recoveries and development (growth and moulting rate) was analysed in a panel of inbred mice (CBA, BALB/c, DBA/2, SJL, 129J, C57BL/10 (B10), C3H/He and NIH), together with mice of BALB and B10 backgrounds with different major histocompatibility complex (H-2) genes (BALB/c, BALB.K, BALB.B and B10, B10.D2/n, B10.BR, B10.S). Parasite recoveries and development were similar in all mouse genotypes tested. They were unaffected by procedures designed to block or modulate phagocytic cell function with carbon or carrageenan, or to suppress inflammation by treatment with hydrocortisone acetate. A comparison of chambers sealed with membranes designed to admit (5·0 μm pore size) or exclude (0·2 μm pore size) host cells demonstrated no effect on the percentage recovery of living larvae, although dead larvae were more frequently retrieved when cells were excluded. Recoveries and rates of development of larvae implanted into immunodeficient scid mice and athymic Hooded rats were similar to those recorded in immunocompetent controls. We conclude that host genetic factors and immunocompetence are not significant determinants of survival, growth or development of O. lienalis larvae implanted within micropore chambers into naive mice. Despite its limitations, the use of this system merits further investigation as an approach to the study of protective immunity against developing larvae in onchocerciasis.

The regulatory role of tumour necrosis factor (TNF) was investigated in murine infection with tetrathyridia of Mesocestoides corti. Recombinant TNFα reduced macrophage larvicidal activity in vitro. M. corti primed mice for TNF release in response to bacterial lipopolysaccharide (LPS) in vivo. TNF activity was amplified 100-fold at 14 days post-infection (p.i.), with a further rise at day 28 p.i. Maximal inflammatory reaction was observed histologically in the liver at the height of TNF activity. Hepatic necrosis was located within inflammatory foci, but not within the vicinity of the parasite itself, suggesting that TNF may contribute to the pathogenesis of infection. Peritoneal cells from infected mice, when stimulated with tetrathyridia in vitro, showed a 4-fold increase in TNFα activity at day 14 p.i. However, when peritoneal cells were stimulated with LPS in vitro, a marked increase in TNFα secretion was observed at 2 months post-infection followed by a slow decline. It is suggested that impaired macrophage effector function, previously attributed to endogenous endotoxin, which gains access to peritoneal macrophages through an inability of the liver to detoxify endotoxin, may be mediated through TNFα.

Two-dimensional polyacrylamide gel electrophoresis was used to examine the differences in total protein composition between two motile stages and two sedentary stages of the potato cyst-nematode, Globodera rostochiensis. Using a sensitive silver stain, 542 reproducible protein spots were distinguished. A list of these spots is presented, showing their apparent molecular weights, estimated isoelectric points, and occurrences in the different developmental stages. When the protein patterns were compared, 401 spots were found to change their presence or size in one or more of the four developmental stages. It is therefore estimated that during the post-embryonic development of G. rostochiensis, 74% of the polypeptides undergo modulation of their synthesis, or are affected by protein degradation or modification. In the motile stages several abundant proteins were present, which disappeared or decreased in concentration in the sedentary stages. Some of these proteins are presumably muscle proteins, and their modulation may illustrate the degeneration of body-wall musculature in the sedentary stages. It is concluded that the potato cyst-nematode has a very dynamic protein metabolism.

Spores of the bacterium Pasteuria penetrans adhered to second-stage juveniles of both Meloidogyne incognita and M. arenaria, but in standard attachment assays far fewer adhered to the latter species. Similarly, a polyclonal antibody was shown not to recognize the surface coat of M. arenaria but did recognize the surface coat of M. incognita. Although the incubation of whole, intact 2nd-stage juveniles of M. incognita in a series of detergents and protein-denaturing agents at room temperature did not reduce the number of spores adhering after exposure to the bacterium, incubation in buffer (PBS) alone at 100 °C for 2 min did. Immunoblotting of cuticle extracts onto nitrocellulose and probing the blots with antibody showed that the antibody recognized a large protein of Mr 80 kDa and a series of smaller proteins of approximately 43 kDa on M. incognita which were not recognized in extracts from M. arenaria. As incubation of the 2nd-stage juveniles in antibody prohibited spore attachment it is suggested that these proteins may be involved in spore adhesion. Large differences exist between M. incognita and M. arenaria in the amounts of surface-associated proteins on the 2nd-stage juvenile cuticle.

The epidemiology of nematode infections of Soay sheep on the island of St Kilda over a period of 2 years (August 1988–August 1990) spanning a host population crash is described. Infective larvae (L3) levels on pasture were high (2422±365 L3/kg D.M. grass in midsummer 1988) when host population density was high, decreasing after the sheep population declined by 70% in early 1989 (601 ±14 L3/kg D.M. in midsummer 1989). The availability of infective larvae to sheep increased during the winter of 1988–1989, probably as a result of concentration of existing larvae on grass as vegetation was destroyed by bad weather and overgrazing. Increased availability of pre-parasitic stages was accompanied by a marked increased in faecal egg counts from sheep of all ages and both sexes. Prevalence and intensity of infection (faecal egg counts) were higher in males than females throughout the 2-year study (χ2 = 208.3, P < 0.005 and F1.2000 = 304, P < 0.001 respectively), except during the lambing periods, and decreased with age in both sexes. Changes in prevalence and intensity of strongyle infections were associated with changes in host population density. Prevalence and intensity of Dictyocaulus filaria larvae in faeces increased during the host population crash. Infection intensity decreased with age (F1.203 = 44.02, P < 0.001) and was higher in males than females (F1.203 = 13.45, P < 0.001).

In early 1989, two-thirds of the Soay sheep population on St Kilda died over 12 weeks. Post-mortem examinations revealed emaciated carcasses and considerable nematode burdens, with protein-energy malnutrition as the probable cause of death. Haematological and blood biochemical changes in the sheep, as well as fecundity of gastrointestinal nematodes, suggested the hosts were immunosuppressed. In parallel, laboratory experiments in which Soay sheep on a high plane of nutrition were artificially infected with Ostertagia circumcincta, showed no clinical signs or mortality when supporting worm burdens similar to those recorded in dead sheep on St Kilda. Anthelmintic treatment of a group of animals increased daily survival rates in ewes and male lambs, although treated animals became re-infected as the ‘crash’ progressed. It is suggested that parasites contribute to mortality in malnourished hosts, exacerbating the effects of food shortage.

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1–39) and to the C-terminal decapeptide of NPF(30–39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.

Potential mechanisms of immunoregulation have been investigated for the capacity to generate heterogeneity in the outcome of infection with helminth parasites. We have developed a mathematical model of the interaction between T cell and parasite populations, based on the assumption that activation of a Th1 CD4+ T cell response is required for host resistance. Antigen dose-dependent inhibition of Th1 cell proliferation generates heterogeneity in the outcome of host response to infection, with relatively low levels of exposure inducing resistance, and high levels of exposure associated with host susceptibility. Heterogeneity is additionally predicted in the duration of infection before individuals of the resistant class clear infection, with infection becoming more prolonged as the level of exposure rises. Similar categories of response are predicted if an alternative regulatory mechanism, that of interferon γ-regulated control of Th1 cell differentiation, is substituted into the model. However, the relationship between level of exposure and duration of infection is reversed. Results are discussed in the context of how these simple models of parasite–immune system interactions might be used to make predictions concerning specific examples of parasitic infection.