F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognized by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

The molecular karyotypes of several different protozoan parasites show high intra-species variation, including different kinetoplastids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp. In this study, the molecular karyotype of Trypanosoma rangeli was examined. To evaluate potential intra-species molecular karyotype variations, 16 different samples were studied by pulsed field gel electrophoresis (PFGE) followed by ethidium bromide staining and hybridizations with 6 different probes. The result showed that different T. rangeli populations are highly polymorphic regarding the molecular karyotype, and thus suggests that PFGE analysis can be used for classification of different T. rangeli isolates. In addition, the molecular karyotype of T. rangeli was compared to molecular karyotypes of other kinetoplastids, and was shown to be distinctly different from that of T. cruzi, but shows some similarities with the karyotype described for T. brucei. Among the probes used one was identified as highly polymorphic, and thus informative for studies of different T. rangeli populations, and another was useful for differentiation between T. rangeli and T. cruzi.

Individual susceptibility to malaria infection, disease and death is influenced by host genotype, parasite virulence and a number of environmental factors including malaria-specific immunity. Immune responses are themselves determined by a combination of host genes and environmental effects. The extent to which host genotype limits the spectrum of possible immune responses may influence the outcome of infection and has consequences for vaccine design. Associations have been observed between human major histocompatibility complex (MHC) genotype and susceptibility to severe malaria, but no similar associations have been observed for mild malarial disease or for specific antibody responses to defined malaria antigens. Epidemiological studies have shown that, in practice, neither T helper cell nor antibody responses to malaria parasites are limited by host MHC genotype, but have revealed that genes lying outside the MHC may influence T cell proliferative responses. These genes have yet to be identified, but possible candidates include T cell receptor (TcR) genes, and genes involved in TcR gene rearrangements. More importantly, perhaps, longitudinal epidemiological studies have shown that the anti-malarial antibody repertoire is selective and becomes fixed in malaria-immune individuals, but is independent of host genotype. These findings suggest that the antibody repertoire may be determined, at least in part, by stochastic events. The first of these is the generation of the T and B cell repertoire, which results from random gene recombinations and somatic mutation and is thus partially independent of germline genes. Secondly, of the profusion of immunogenic peptides which are processed and presented by antigen presenting cells, a few will, by chance, interact with T and B cell surface antigen receptors of particularly high affinity. These T and B cell clones will be selected, will expand and may come to dominate the immune response, preventing the recognition of variant epitopes presented by subsequent infections - a process known as original antigenic sin or clonal imprinting. The immune response of an individual thus reflects the balance between genetic and stochastic effects. This may have important consequences for subunit vaccine development.

The radiation-attenuated schistosome vaccine induces a high level of protective immunity in rodents. In order to assess its potential relevance to man, we have tested its efficacy in the non-human primate Papio anubis. A vaccination regime consisting of 3 exposures of approximately 9000 cercariae irradiated with 30 or 60krad. of gamma radiation induced > 50% protection to a challenge with normal larvae. A lower attenuating dose of 20 krad., optimal for vaccination of mice, was less effective. All vaccination regimes elicited a population of PBMC which proliferated in vitro in response to antigen. These responses peaked after the third exposure but were significantly lower after challenge. They revealed relatively little cross-reactivity with adult Schistosoma haematobium antigens and provided some evidence for stage-specific antigens. Circulating IgM reactive with adult S. mansoni antigen was detected after the second vaccination but levels remained low throughout. In contrast, IgG levels were boosted by successive vaccinations, although they showed a tendency to decline from 14 days after each exposure. There also appeared to be a lag of about 14 days after challenge before levels began to rise. Thus, both proliferation and antibody data suggest a lower responsiveness after challenge which may reflect either the reduced antigenic load or immunogenicity of normal, compared to vaccinating larvae. The data indicate that the attenuated schistosome vaccine is capable of inducing protection in a highly permissive primate host, with the implication that the mechanisms involved may also be relevant to man.

Paramyosin, although a widely distributed muscle component among invertebrates, has hitherto not clearly been shown to occur in the muscles of schistosomes. Instead, it has been reported to occur in the tegument. In the present study, a specific antibody reacting with each of 10 isoforms of paramyosin was used for light microscopical immunolocalization in sections of Schistosoma mansoni. Specimens were fixed by a new method to immobilize antigens with uranyl acetate–trehalose–methanol. In cercariae, schistosomula, and adults, the circular and longitudinal muscles of the body wall, the dorsoventral muscles and those surrounding the gut and the pharynx as well as the fast moving cross-striated muscles of the tail of cercariae intensely reacted with the antibody. However, neither immunohistologically nor on Western blots of isolated tegument, were indications found for the presence of paramyosin in the tegument. In vivo phosphorylation and binding of anti-phospho-tyrosine and anti-phospho-serine antibodies show phosphorylation of paramyosin which probably is responsible for the generation of the isoforms.

Neospora caninum and N. caninum-like organisms are cyst-forming coccidian parasites known to cause neuromuscular disorders in dogs and abortion in cattle. In this article we report on the use of the polymerase chain reaction (PCR) for the detection of DNA from N. caninum. After determining the sequence of the internal transcribed spacer 1 (ITSl) of N. caninum and Toxoplasma gondii, and part of the sequences for & species of Sarcocystis, we designed a primer set for the amplification of a 279-base-pair fragment of ITSl from N. caninum. The PCR system made possible the specific detection of 5 N. caninum organisms and no amplification was observed from any of the other cyst-forming coccidia tested, including the closely related T. gondii. Furthermore, we were also able to demonstrate the presence of N. caninum in brain and lung tissue samples from experimentally infected mice. Our data also link the 5-8S rRNA gene for T. gondii and N. caninum to the 16S-like rRNA gene, within the rDNA unit.

The detection of egg-derived antigens in the serum and urine of Schistosoma mansoni-infected individuals and experimental animals would provide an alternative method to assess the tissue egg burden. The detected levels are, however, not only a function of the amounts of antigen produced, but also of the processing or clearance by the host. In the present study the immunolocalization pattern of antigens using 2 recently described monoclonal antibodies to repetitive carbohydrate epitopes of S. mansoni soluble egg antigen (114–5B1–A and 114–4D12–A) in various organs of the host was investigated. In the liver strong immunoreactivity could be detected around the entrapped eggs and in egg-shells, as well as in Kupffer cells accumulating both antigen and schistosomal pigment. In the spleen, immunohistochemistry revealed antigen in the plasma as well as in secondary lysosomes of macrophages. Strong labelling was found in the vesicles of the eosinophilic granulocytes: indirect evidence perhaps for the presence of antigen–antibody complexes. In conclusion, the secreted egg antigens were sequestered in the reticulo-endothelial macrophages of the liver and the spleen as already partly described for worm-derived antigens. The presence of large quantities of antigenic material in the spleen could suggest an important role of this organ in the clearance of antigen and might even provide an additional explanation for the hepatosplenomegaly mainly present in S. mansoni-infected children.

A sensitive fluorometric method for assaying malarial pigment, haemozoin, has been developed and used to determine the haemozoin content of blood and tissue samples. Plasmodium falciparum rings and trophozoites were found to contain 23 and 339 ng haemozoin/106 parasitized red blood cells (PRBCs), respectively. Unsynchronized Plasmodium berghei NK65 or ANKA parasites from infected mice contained 27 and 61 ng haemozoin/106 PRBCs, respectively. An exponential accumulation of haemozoin within 18 days after infection was demonstrated in liver and spleen tissue, representing up to 0·2% of the tissue by wet weight by day 18. Histology indicated that the accumulation occurred predominantly in the tissue monocytes. In the brain, the levels of haemozoin after 8 days of infection were considerably lower than they were in the liver or spleen, and most of the pigment appeared to be that present inside parasitized red blood cells. CBA/Ca mice infected with P. berghei ANKA (a cerebral malaria model) had significantly higher amounts of haemozoin in the brain than did ICR mice infected with P. berghei NK65. Thus, haemozoin levels in tissue increase with the duration of infection, and its presence may be associated with cerebral pathology.

Neospora caninum is a recently identified coccidian parasite which was, until 1988, misdiagnosed as Toxoplasma gondii. It causes paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. The life-cycle of Neospora has not yet been elucidated. The only two stages identified so far are tissue cysts and intracellularly dividing tachyzoites. Very little is known about the biology of this species. We have set up a fluorescence-based adhesion/invasion assay in order to investigate the interaction of N. caninum tachyzoites with bovine aorta endothelial (BAE) cells in vitro. Treatment of both host cells and parasites with metabolic inhibitors determined the metabolic requirements for adhesion and invasion. Chemical and enzymatic modifications of parasite and endothelial cell surfaces were used in order to obtain information on the nature of cell surface components responsible for the interaction between parasite and host. Electron microscopical investigations defined the ultrastructural characteristics of the adhesion and invasion process, and provided information on the intracellular development of the parasites.

Based on the ability of bacterial associates of entomopathogenic nematodes to produce antibiotic compounds on artificial media, it has been commonly accepted that Xenorhabdus sp. and Photorhabdus sp. inhibit a wide range of invading microorganisms in insects infected with Steinernema spp. or Heterorhabditis spp. Therefore, the question of whether antibiotic compounds produced by the primary form of bacterial symbionts associated mutualistically with S. carpocapsae and H. bacteriophora explain why insect carcasses do not putrefy but provide nutritional requirements for insect parasitic rhabditoid nematodes to complete their life-cycle was examined. Laboratory bioassays of anti-bacterial activity on nutrient agar and during parasitism in larvae of Galleria mellonella have confirmed earlier observations that in vitro colonies of the primary form of X. nematophilus and P. luminescens produced agar-diffusible antibiotic compounds of a broad spectrum of anti-bacterial activity; their role in parasitism seems doubtful, however. This hypothesis is supported by a low antibiotic potency of a limited spectrum of anti-bacterial activity throughout the life-cycle of the parasites, principally in Galleria infected with S. carpocapsae. Since the lack of putrefaction cannot be explained simply by antibiotic inhibition of contaminating bacterial microflora, other competition mechanisms must be operating in parasitized insects. I postulated that a rapid and massive colonization of the insect body by nematophilic bacteria creates unfavourable conditions for the growth and multiplication of bacterial (proteolytic) contaminators making the insect carcass decay-resistant. In the case of H. bacteriophora, low antibiotic activity at an early stage of parasitism could support the colonization by P. luminescens of the host.

The neutral glycolipid fraction from mouse-propagated, Schistosoma mansoni adult worms has been investigated as to its chromatographic and antigenic properties, and whether it fulfills the serodiagnostic antigen requirements of sensitivity and specificity in the detection of schistosomiasis. Serological analyses were performed by thin-layer chromatography immunostaining and ELISA. In the acute-phase form of mouse schistosomiasis, the kinetics of development of neutral glycolipid-specific antibody levels was correlated with the intensity of the initial infection and the response was dominated by IgG, as represented by the subclass IgG1. With the experimental animal helminthiases screened, glycolipid antigenicity fulfilled the fundamental traits for a serodiagnostic reagent. In the chronic-phase form of human schistosomiasis mansoni, neutral glycolipid-specific antibody levels were not correlated with the intensity of infection, as estimated from the faecal content of parasite eggs, whilst the isotypic response was dominated by IgM and IgG, the latter represented primarily by IgG1 and secondarily by IgG3. With other human helminthiases, glycolipid antigenicity was incomplete, in that, the specificity was only partially fulfilled. The reason for this incomplete specificity has been clarified, in part, by the detection of cryptic schistosomiasis infections in the cohorts of African patient sera examined.

A temporal study of the effects on the tegument of Schistosoma mansoni adult worm following in vivo praziquantel and oxamniquine treatment was performed. Drug-induced damage to the tegument, exposure of surface antigens and attachment of host antibody occurred rapidly, within 1 h, following praziquantel treatment. Oxamniquine-treated worms required 4–8 days for these effects to be apparent. The 2 drugs differed in the degree and sites of damage on the worm surface. The administration of 2 different polyspecific rabbit sera with drug significantly increased the efficacy of praziquantel when administered with the drug, but not when given 6–9 days after drug treatment. In contrast, only 1 serum was synergistic with oxamniquine when administered with drug and both sera were synergistic when given 6–9 days after drug treatment. The effect of immune killing of drug-treated worms is discussed.

Expression of metalloprotease activities during metacyclogenesis of a series of strains and clones of Trypanosoma cruzi was investigated using SDS-polyacrylamide gel electrophoresis with a range of different co-polymerized proteolytic substrates. A complex pattern of metalloprotease expression, with considerable quantitative and qualitative variability between strains and life-cycle stages, was apparent. This is in contrast to previously published data, and data presented in this study, which demonstrate a high degree of conservation of expression of cysteine proteases in different strains and clones of T. cruzi. All the metalloprotease activities identified partitioned into the detergent phase of a Triton X-114 extract, suggesting that they are membrane-bound. Developmental regulation of expression during metacyclogenesis, either in terms of quantity, or in terms of difference in relative amounts of different isoforms, was apparent for all isolates studied except CL14. However, a clearly metacyclic-specific/metacyclic-enriched metalloprotease was detected only in T. cruzi Dm28c and 383, and our results demonstrate that a metacyclic-specific metalloprotease common to all isolates of the parasite could not be detected, at least at this level of analysis.

Carbohydrates associated with the tegumental surface of Schistosoma mansoni primary sporocyst may serve as potential receptors for mediating recognition by the internal defence system of the molluscan host, Biomphalaria glabrata. Therefore, a combination of SDS-PAGE and lectin probe analyses were carried out on biotin-labelled tegumental glycopeptides as a first step to defining the carbohydrates expressed at the sporocyst surface. The majority of surface polypeptides, ranging in relative molecular masses from 27 to 113 kDa, reacted with horseradish peroxidase-labelled Canavalia ensiformis (Con A), Erythrina corallodendron (ECA), Glycine max (SBA) and Triticum vulgaris (WGA) lectins indicating that most, if not all, tegumental proteins are glycosylated. However, differences in the binding of some lectins to individual glycopeptides suggest a degree of heterogeneity in the structure/composition of sugar moieties comprising these surface glycoconjugates. This notion is supported by the finding that the fucose-specific Tetragonolobus purpureas (TPA) lectin only reacted with approximately 50% of glycopeptides identified at the tegumental surface. Experiments employing biotin-labelled plasma (cell-free haemolymph) from S. mansoni-susceptible and -resistant B. glabrata snails as probes, further demonstrated that many of the identified surface glycoproteins also serve as plasma-binding sites for both snail strains. Binding interactions between plasma and sporocyst surface glycoproteins appeared to be, at least in part, mediated by carbohydrates since periodate treatment of sporocyst proteins or pre-incubation of plasma with the glycoproteins, fetuin or mucin, resulted in a decrease in plasma reactivity to blotted larval proteins.

Infections with Theileria parva in the African buffalo are invariably asymptomatic, whereas infections in cattle usually result in clinical disease, the severity of which varies in different populations of cattle. The parasite exhibits antigenic heterogeneity, which in cattle manifests as differences between parasite strains in their cross-protective properties. A series of studies on T cell responses to T. parva in cattle have demonstrated that class I MHC-restricted cytotoxic T lymphocytes (CTL), specific for parasitized lymphoblasts, are important mediators of immunity. Cytotoxic T cell responses frequently display parasite strain-restricted specificities which appear to correlate with the capacity of strains to cross-protect. The strain specificity of CTL responses varies in animals immunized with the same parasite strain and is influenced by both host and parasite genotype. Recent studies have provided evidence that there is competition between epitopes for induction of CTL responses, which can result in a bias to strain-specific epitopes. These properties of the CTL response have important implications for vaccination. Thus, in designing a vaccine, it may be possible, by selecting parasite proteins containing appropriate CTL epitopes, to generate CTL responses that protect against a wide range of parasite strains. Although there are no comparable data on CTL responses in the buffalo, it is considered that the features of the immune response described for cattle would be advantageous for survival of parasite populations in the buffalo. Specifically, a bias in the immune responses to strain-specific determinants should favour establishment of infection in buffalo already carrying the parasite and allow fluctuation in the levels of different parasite strains during the course of persistent infection.

Quinacrine resistance has been induced in 3 Giardia duodenalis laboratory stocks and 4 lines resistant to other drugs. The quinacrine-resistant lines were maintained in normally lethal levels of 5 µM quinacrine and some lines are viable in 20 µM levels of the drug. Fluorescence studies indicated that quinacrine was taken up by sensitive cells but was actively excluded from resistant trophozoites. The nuclei were not a site of drug accumulation and no specific fluorescence in the trophozoite could be attributed to any structure. Blebs of concentrated drug appeared prior to disintegration of the membrane of drug-sensitive trophozoites exposed to drug overnight. Parasite lines already resistant to furazolidone adapted more readily to quinacrine exposure than drug-sensitive stocks. This multiple drug resistant phenotype was not as marked with metronidazole-resistant lines.

Two important recent advances in Leishmania immunology are: (i) the demonstration of a dramatic dichotomy in T helper 1 versus T helper 2 subset expansion leading to protection versus disease exacerbation; and (ii) analysis of the macrophage activation pathways leading to enhanced intracellular killing of parasites, in particular the tumour necrosis factor α (TNFα)-dependent sustained induction of the inducible nitric oxide synthase gene (Nos2) leading to the generation of large amounts of nitric oxide (NO). Given the broad spectrum of disease phenotypes in human leishmaniasis, one might predict that a genetic defect at any key point in this macrophage activation pathway and/or in pathways leading to activation of different T cell subsets, and the latter may be a pleiotropic effect of the former, will contribute to disease susceptibility. By studying disease in genetically-defined inbred mouse strains, it has been possible to identify 5 regions of the murine genome carrying leishmanial susceptibility genes. The genes include: (i) Scl-2 (mouse chromosme 4/human chromosome 9p; candidate Janus tyrosine kinase 1) controlling a unique no lesion growth resistance phenotype to Leishmania mexicana; (ii) Scl-1 (distal mouse chromosome 11/human 17q; candidates Nos2, Sigje, MIP1α, MIP1β) controlling healing versus non-healing responses to L. major; (iii) the ‘T helper 2’ cytokine gene cluster (proximal murine chromosome 11/human 5p; candidates IL4,5,9) controlling later phases of L. major infection; (iv) the major histocompatibility complex (MHC: H-2 in mouse, HLA in man: mouse chromosome 17/human 6p; candidates class II and class III including TNFα/β genes); and (v) Nramp1, the positionally cloned candidate for the murine macrophage resistance gene Ity/Lsh/Bcg (mouse chromosome 1/human 2q35). This review examines these 5 regions and the candidate genes within them, reflecting on their current status as candidates for human disease susceptibility genes.

Acetylcholinesterase (ACHE) is present in the muscle and on the tegument of schistosomes. Molecular forms of schistosome AChE were examined because particular AChEs are found in tissues of distinct function elsewhere. The dimeric globular form (G2) is the only form evident in adult Schistosoma haematobium: 32 % of the muscle AChE is hydrophilic and 61 % is membrane associated. A substantial amount of this enzyme is phosphatidylinositol (PI) anchored since it could be released by PI-specific phospholipase C from both muscle and tegumental membranes.

Polymerase chain reaction (PCR) combined with non-radioactive DNA hybridization was applied for the detection and characterization of a 150 bp tandem repeat of Onchocerca volvulus. DNA of worms from western Uganda was amplified and then probed with a digoxygenin-labelled oligonucleotide, specific for the forest form of O. volvulus and compared to samples from various African countries. Hybridization was only observed with PCR products from the forest in Liberia, south-eastern Ghana, Benin and southern Cameroon, but not with worms from Uganda or the savannah in Burkina Faso and northern Ghana. A nested PCR using primers derived form the forest form-specific DNA sequence confirmed these results. Morphometric studies revealed length differences between the microfilariae of Ugandan O. volvulus to those of West Africa, especially to those of the savannah in Burkina Faso. It is concluded that the forest/savannah classification of O. volvulus from West Africa is not suitable for Simulium weasel-transmitted O. volvulus from Uganda.

The Index of Association (IA) has been proposed by Maynard Smith et al. (1993) as a general method for characterizing the population structures of microorganisms as either: clonal, epidemic, cryptic species or panmictic. With reference to the current debate surrounding the mode of reproduction in parasitic protozoa, this study explores (i) the suitability and limitations of the IA for characterizing populations of Trypanosoma brucei s.l., and (ii) the idea that the significance of genetic differences between populations may be better understood if the evolution, spread and temporal stability of certain parasite genotypes are also considered. Four populations of T. brucei from Côte d'Ivoire, Uganda and Zambia are analysed using the IA and a complementary test for linkage disequilibrium, test f of Tibayrenc, Kjellberg & Ayala (1990). The two populations from Uganda are characterized as epidemic, while the others appear more or less clonal; the merits of the two methods are compared. The implications of the various population classifications are discussed with reference to genotype longevity in each region; the evolutionary and biomedical consequences of the genetic non-homogeneity of T. brucei are reviewed.