Molecular approaches are providing new insights into the genetic diversity of schistosomes and their intermediate snail hosts. For instance, molecular tools based on the polymerase chain reaction are being developed for the diagnosis of schistosomiasis and the detection of prepatent schistosome infections in snails at transmission sites. Robust phylogenies of the different species of Schistosoma, Bulinus and Biomphalaria have been determined and novel methods are available to identify the different and cryptic taxa involved. Microsatellite analyses and mitochondrial DNA sequencing methods have been developed and are contributing to a better understanding of the genetic structure of both schistosome and snail populations. New sampling procedures to capture DNA of eggs and larval stages of schistosomes in field situations are facilitating more detailed and ethically advantageous studies on parasite heterogeneity. Knowledge of the genetic diversity of schistosome and snail populations adds a further dimension to the monitoring and surveillance of disease, and the implementation of new molecular-based approaches will be of increasing importance in helping to assess the impact of schistosomiasis control strategies.

Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.

Ectoparasites of livestock are of great economic and social importance but their effective control remains difficult. The feasibility of vaccination as a novel control measure was established over a decade ago with the commercial release of a recombinant vaccine against the cattle tick Boophilus microplus. Since then, research has continued on ticks and other ectoparasites. While some ectoparasite species will undoubtedly be refractory to immunological control, for others there has been a steady accumulation of knowledge of partially protective antigens, now accelerating through the application of genomic technologies. Nevertheless, progress towards usable, commercially available vaccines has been limited by a number of factors. The number of highly effective antigens is still very small. Although some classes of antigen have been investigated in more detail than others, we have no systematic knowledge of what distinguishes an effective antigen. Much hope has been placed on the potential of multi-antigen mixtures to deliver the efficacy required of a successful vaccine but with little experimental evidence. The application of current knowledge across parasite and host species needs to be explored but little has been done. In most cases, the path to commercial delivery is uncertain. Although many constraints and challenges remain, the need for vaccines and our capacity to develop them can only increase.

Host immune responses have been shown to enhance the efficacy of several schistosomicidal drugs. The evidence derives mainly from experiments on Schistosoma mansoni infections in the mouse with their immune status variously modulated; this review emphasises praziquantel (PZQ), which is now the main drug used for treatment of human schistosomiasis. Electron microscopy and indirect immunofluorescence indicate that PZQ disrupts the integrity of the surface membranes of S. mansoni, particularly those covering the dorsal tubercles of adult male worms, and this causes antigens which are the targets of antibody attack to be revealed. We review the evidence that two S. mansoni antigens in particular are implicated in the immune-dependent action of PZQ: a 200 kDa glycoprotein and a 27 kDa antigen with non-specific esterase activity.

The interaction between mouse peritoneal macrophages and ‘Y’ strain Trypanosoma cruzi bloodstream forms was studied at optical and electron microscopical levels. The method of marking lysosomes with Thorotrast, either before or after infection of cell monolayers with parasites, revealed that secondary lysosomes fused with phagosomes shortly after trypanosome interiorization. In spite of this, 24 h later most parasites were no longer in a vacuole but lay free within the host cell cytoplasm, multiplying actively. At this time, and up to shortly before 96 h when parasites escaped to the external milieu, most parasitized cells were not lethally injured, as revealed by the Trypan blue dye-exclusion test. Only when parasites were released into the external medium was this situation reversed and infected macrophages took up the dye.

A gene, CRP136, from the intestinal protozoan parasite Giardia duodenalis, expressed at a high level in a metronidazole-resistant line, encodes a 136 kDa protein with 23 copies of a 40 amino acid repeat. The protein is cysteine-rich and has the typical membrane-spanning region and CXXC amino acid motifs of a family of Giardia cysteine-rich surface proteins (CRSPs). The repeat unit in CRP136, shares 57% homology with the gene encoding the precursor of the sarafotoxins, a group of snake toxins from the burrowing adder known to cause symptoms similar to those of humans acutely infected with Giardia. The sarafotoxins are low molecular weight sulphydryl cross-linked peptides which are proteolytically cleaved from a precursor polyprotein. CRP136 has homology over the entire length of the sarafotoxin precursor, and the repeats are of the same length. Thus CRP136 represents the first evidence for a potential Giardia toxin. The genomic copy number of CRP136 appears to be the same in both the parent and drug-resistant lines and expression of this gene, and at least one other, is associated with a conserved partial duplication, but not amplification, of one chromosome.

Plasmodium mexicanum is a common malarial parasite of the western fence lizard, Sceloporus occidentalis, in northern California, USA. Infected female lizards store substantially less fat during the summer activity season and produce smaller clutches of eggs than do non-infected animals. Stored fat is utilized in the production of eggs; the energy content of the decrement in stored fat is approximately equal to the energy content of the average reduction in number of eggs. Thus, there is ongoing strong selective pressure on the host to evolve appropriate anti-parasite measures.

Blood digestion was studied in strains of Anopheles stephensi which had been genetically selected for either refractoriness or susceptibility to infection by Plasmodium falciparum. Females of the refractory Pb3—9a strain ingested more blood than selected (Sda-500) and unselected (Punjab) susceptible females and began to degrade the haemoglobin soon after feeding. In susceptible females, haemoglobin degradation started only after a significant post-feeding lag period. Total protein content of the midgut after the bloodmeal was correspondingly higher for refractory than for susceptible females, but absolute and relative rates of protein degradation were not significantly different between the different mosquito strains. Bloodmeal induction of midgut trypsin activity and the maximal trypsin activity were the same for the different strains. The residual aminopeptidase activity and its relative post-feeding activity (enzyme units per midgut) were significantly higher in refractory females. However, when converting to specific aminopeptidase activity, no differences between strains were evident. The results indicate that both the early initiation of haemoglobin degradation and higher aminopeptidase activity in the Pb3—9a refractory strain are important in the limitation of parasite development within the mosquito midgut, whereas trypsin plays no role in this process.

The epidemiology of Haemonchus contortus was studied in north east England in lambs reared under field conditions with infected ewes. The parasite population was monitored by pasture larval counts, faecal egg counts and lamb postmortems. From 25 June to 11 November, eight lambs were slaughtered each month, together with eight worm-free tracer lambs. Postmortem worm counts showed that on 25 June, small numbers of adult H. contortus were present but no inhibited larvae. However, subsequently the number of worms recovered rose rapidly to 1000–5000 and the percentage inhibition increased to 57% in July, 75% in August and virtually 100% in September, October and November. On each sampling occasion no significant differences in worm count or percentage inhibition were recorded between experimental and tracer lambs. These results suggest that neither autumn climatic effects nor host immunity were responsible for inhibition in this strain of H. contortus.

Larval helminths often share individual intermediate hosts with other larval worms of the same or different species. In the case of immature acanthocephalans capable of altering the phenotype of their intermediate hosts, the benefits or costs of host sharing can be evaluated in terms of increased or decreased probability of transmission to a suitable definitive host. Competitive interactions among the immature stages of acanthocephalans within the intermediate host could create additional costs of host sharing, however. The effects of intraspecific and interspecific interactions were measured in 3 sympatric species of acanthocephalans exploiting a population of the amphipod Echinogammarus stammeri in the River Brenta, Italy. The strength of interactions was assessed from differences in the size achieved by infective cystacanths in the intermediate host. The size of Pomphorhynchus laevis cystacanths was not correlated with host size, whereas the size of Acanthocephalus clavula and Polymorphus minutus cystacanths increased with host size. Reductions in cystacanth size caused by intraspecific competition were only detected in P. laevis, but may also occur in both A. clavula and P. minutus. When co-occurring in the same amphipod with cystacanths of A. clavula, cystacanths of P. laevis attained a smaller size than when they occurred on their own. This effect was not reciprocal, with the size of A. clavula cystacanths not being affected. This supports earlier suggestions that it is adaptive for A. clavula to associate with P. laevis in amphipod intermediate hosts, with both species going to the same fish definitive hosts. In contrast, cystacanths of P. laevis achieved their largest size when they co-occurred in an amphipod with a cystacanth of P. minutus, which has a different definitive host (i.e. birds). These findings suggest that the net benefits of sharing an intermediate host can only be estimated by taking into account both the effects on transmission success and the consequences for cystacanth development.

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

The survival characteristics of Discocotyle sagittata larvae are both age- and temperature-dependent. In laboratory studies at constant temperatures of 6, 10, 13, 18 and 22°C, oncomiracidia had a maximum life-span of 96 h at 6°C, declining with increasing temperature to 26 h at 22°C. Larval swimming activity was also age dependent, and continued for a large proportion of survival time. The relationship between time at which larvae stop swimming as a proportion of total survival time was statistically significant between temperatures. Proportionally, oncomiracidia swam for longer periods at lower temperatures. As oncomiracidia age, they become progressively less active, spending less time in the water column. In contrast to observations recorded in the literature, D. sagittata larvae are infective soon after emergence (within the minimum of 2 h studied), and feed on blood from the gill capillaries within 2 h post-exposure. The temperature-dependent characteristics of the larval stage are likely to have important implications for seasonal changes in parasite transmission.

The proteolytic activity of extracts of Entamoeba histolytica has been further investigated. Casein and gelatin, but not haemaglobin, were hydrolysed. Activity was observed in the pH range 5·8 to 8·5 with optimal activity at pH 7·5 to 7·9. Activity was optimal at 37° C. and sulphydryl groups were not required. Concomitant bacteria showed no proteolytic activity. Hyaluronidase, collagenase and lecithinase could not be detected.

An inhibitor of proteolytic enzyme was present in sera of all animals tested and in egg yolk. All culture media prepared from eggs were inhibitory, but inspissation or dilution of serum inactivated the serum inhibitor. Purified trypsin inhibitors from lima and soy bean were not active against the amoebic enzyme. Proteolytic enzymes were not secreted extracellularly in vitro.

High proteolytic activity was found in two out of five invasive, freshly isolated, strains of E. histolytica and after two series of liver passages of a single strain. The significance of these observations is discussed. It is concluded that the present evidence does not convincingly demonstrate that high proteolytic activity is required for tissue invasion by amoebae, but may accompany another factor.

We present a simple unscaled, quantitative framework that addresses the optimum use of resources throughout a host's lifetime based on continuous exposure to parasites (rather than evolutionary, genetically explicit trade-offs). The principal assumptions are that a host's investment of resources in growth increases its survival and reproduction, and that increasing parasite burden reduces survival. The host reproductive value is maximised for a given combination of rates of parasite exposure, host resource acquisition and pathogenicity, which results in an optimum parasite burden (for the host). Generally, results indicate that the optimum resource allocation is to tolerate some parasite infection. The lower the resource acquisition, the lower the proportion of resources that should be devoted to immunity, i.e. the higher the optimum parasite burden. Increases in pathogenicity result in reduced optimum parasite burdens, whereas increases in exposure result in increasing optimum parasite burdens. Simultaneous variation in resource acquisition, pathogenicity and exposure within a community of hosts results in overdispersed parasite burdens, with the degree of heterogeneity decreasing as mean burden increases. The relationships between host condition and parasite burden are complicated, and could potentially confound data analysis. Finally, the value of this approach for explaining epidemiological patterns, immunological processes and the possibilities for further work are discussed.

Recombinant NcPDI(recNcPDI), NcROP2(recNcROP2), and NcMAG1(recNcMAG1) were expressed in Escherichia coli and purified, and evaluated as potential vaccine candidates by employing the C57Bl/6 mouse cerebral infection model. Intraperitoneal application of these proteins suspended in saponin adjuvants lead to protection against disease in 50% and 70% of mice vaccinated with recNcMAG1 and recNcROP2, respectively, while only 20% of mice vaccinated with recNcPDI remained without clinical signs. In contrast, a 90% protection rate was achieved following intra-nasal vaccination with recNcPDI emulsified in cholera toxin. Only 1 mouse vaccinated intra-nasally with recNcMAG1 survived the challenge infection, and protection achieved with intra-nasally applied recNcROP2 was at 60%. Determination of cerebral parasite burdens by real-time PCR showed that these were significantly reduced only in recNcROP2-vaccinated animals (following intraperitoneal and intra-nasal application) and in recNcPDI-vaccinated mice (intra-nasal application only). Quantification of viable tachyzoites in brain tissue of intra-nasally vaccinated mice showed that immunization with recNcPDI resulted in significantly decreased numbers of live parasites. These data show that, besides the nature of the antigen, the protective effect of vaccination also depends largely on the route of antigen delivery. In the case of recNcPDI, the intra-nasal route provides a platform to generate a highly protective immune response.

Gastrointestinal (GI) nematodes are important disease-causing organisms, controlled primarily through treatment with synthetic drugs, but the efficacy of these drugs has declined due to widespread resistance, and hence new drugs, with different modes of action, are required. Some medicinal plants, used traditionally for the treatment of worm infections, contain cysteine proteinases known to damage worms irreversibly in vitro. Here we (i) confirm that papaya latex has marked efficacy in vivo against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, (ii) demonstrate the dose-dependent nature of the activity (>90% reduction in egg output and 80% reduction in worm burden at the highest active enzyme concentration of 133 nmol), (iii) establish unequivocally that it is the cysteine proteinases that are the active principles in vivo (complete inhibition of enzyme activity when pre-incubated with the cysteine proteinase-specific inhibitor, E-64) and (iv) show that activity is confined to worms that are in the intestinal lumen. The mechanism of action was distinct from all current synthetic anthelmintics, and was the same as that in vitro, with the enzymes attacking and digesting the protective cuticle. Treatment had no detectable side-effects on immune cell numbers in the mucosa (there was no difference in the numbers of mast cells and goblet cells between the treated groups) and mucosal architecture (length of intestinal villi). Only the infected and untreated mice had much shorter villi than the other 3 groups, which was a consequence of infection and not treatment. Plant-derived cysteine proteinases are therefore prime candidates for development as novel drugs for the treatment of GI nematode infections.

During trans-dermal invasion of the vertebrate host, larval schistosomes (cercariae) transform into schistosomula and become enveloped by a double lipid bilayered, tegumental membrane. The glucose transporter protein SGTP4 is found exclusively in these host-interactive tegumental membranes and in membranous vesicles proposed to be their precursor. In this study, we monitored the appearance and migration of this tegumental marker protein during larval transformation to test the current model of tegumental membrane biosynthesis in parasitic blood flukes. Only minutes after transformation was initiated, SGTP4 began accumulating in a previously unrecognized, bilaterally symmetrical, ‘cyton network’ beneath the peripheral muscle. Approximately 30min after the initiation of transformation the marker protein was seen in tubules connecting the network to the surface and erupting onto the surface in discrete patches. After 1h the patches were regularly arrayed over the schistosomula body and began to cover the anterior organ. By 3h the staining has largely resolved into a contiguous layer of fluorescence covering most of the worm surface. These findings confirm earlier suggestions, based on electron microscopy, that the parasite's surface tegumental membranes are derived from the migration of membranous vesicles produced within cytons and reveal a new subtegumental architecture interconnecting the cytons.

The daily food intake and related changes in body weight were measured in young rats which were fed on synthetic diets containing 1% (3 experiments), 2% (4 experiments) and 20% (1 experiment) casein respectively. Some rats were infected with Nippostrongylus brasiliensis and allowed to feed ad libitum, some remained uninfected and were allowed to feed ad libitum while others, which were matched by initial weight with rats from the infected group, remained uninfected and were given only the same amount of food as that consumed during the previous 24 h by their infected partners. No significant differences were detected between the rats before the start of the infection. Thereafter, the food intake of the infected undernourished rats was usually found to be significantly lower and the loss of body weight significantly greater than those of the uninfected rats. In infection periods of relatively long duration, the pair-fed uninfected rats did not lose as much weight as their infected partners even though their food intake (1% and 2% casein) was the same. Although the food intake, and consequently the weight, of infected rats fed on the diet containing 20% casein were significantly less than in their uninfected partners, no significant differences were observed between the weights of the infected and pair-fed rats fed on this diet.

Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals worldwide. However, information from eastern European countries is sketchy. In many eastern European countries, including Romania, it has been assumed that chronic T. gondii infection is a common cause of infertility and abortion. For this reason, many women in Romania with these problems were needlessly tested for T. gondii infection. Most papers on toxoplasmosis in Romania were published in Romanian in local journals and often not available to scientists in other countries. Currently, the rate of congenital infection in Romania is largely unknown. In addition, there is little information on genetic characteristics of T. gondii or prevalence in animals and humans in Romania. In the present paper we review prevalence, clinical spectrum and epidemiology of T. gondii in humans and animals in Romania. This knowledge should be useful to biologists, public health workers, veterinarians and physicians.

The merozoite surface antigens of malaria parasites are prime anti-morbidity/mortality vaccine candidates. However, their highly polymorphic nature requires extensive surveys of parasite populations to validate vaccine designs. Previous studies have found 3 molecular types (A, B and C) of the Plasmodium vivax merozoite surface protein 3α (PvMSP-3α) among parasite field populations. Here we analysed complete PvMSP-3α sequences from 17 clinical P. vivax isolates from Thailand and found that the nucleotide diversity was as high as that from samples widely separated by time and space. The polymorphic sites were not randomly distributed but concentrated in the N-terminal Ala-rich domain (block 2A), which is partially deleted in type B and C sequences. The size variations among type A sequences were due to small indels occurring in block 2A, whereas type B and C sequences were uniform in length with each type having a different large deletion. Analysis of synonymous and non-synonymous substitutions suggested that different selection forces were operating on different regions of the molecule. The numerous recombination sites detected within the Ala-rich domain suggested that intragenic recombination was at least partially responsible for the observed genetic diversity of the PvMSP-3α gene. Phylogenetic analysis failed to link any alleles to a specific geographical origin, even when different domains of PvMSP-3α were used for analysis. The highly polymorphic nature and lack of geographical clustering of isolates suggest that more systematic investigations of the PvMSP-3α gene are needed to explore its evolution and vaccine potential.