Triclabendazole (TCBZ) is a halogenated benzimidazole compound that possesses high activity against immature and adult stages of the liver fluke, Fasciola hepatica. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the development of liver flukes resistant to this compound. TCBZ sulphoxide (TCBZSO) and TCBZ sulphone (TCBZSO2) are the main molecules recovered in the bloodstream of TCBZ-treated animals. In order to gain some insight into the possible mechanisms of resistance to TCBZ, the goals of the work described here were: to compare the ex vivo transtegumental diffusion of TCBZ parent drug and its sulpho-metabolites (TCBZSO and TCBZSO2) into TCBZ-susceptible and -resistant liver flukes; and to assess the comparative pattern of TCBZ biotransformation by TCBZ-susceptible and -resistant F. hepatica. For the tegumental diffusion studies, TCBZ-susceptible (Cullompton) and -resistant (Sligo) adult flukes collected from untreated infected sheep were incubated (15–180 min) in KRT buffer containing either TCBZ, TCBZSO or TCBZSO2 (5 nmol.ml−1). For the metabolism studies, microsomal fractions obtained from TCBZ-susceptible and -resistant flukes were incubated for 60 min with TCBZ (40 μM), and the amount of the formed metabolic product (TCBZSO) was measured. Drug/metabolite concentrations were quantified by HPLC. All the assayed TCBZ-related molecules penetrated through the tegument of both TCBZ-susceptible and -resistant flukes. However, significantly lower (approximately 50%) concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes compared to the TCBZ-susceptible ones over the 180 min incubation period. The rate of TCBZ sulphoxidative metabolism into TCBZSO was significantly higher (39%) in TCBZ-resistant flukes. The flavin-monooxigenase (FMO) enzyme system appears to be the main metabolic pathway involved in the formation of TCBZSO in both TCBZ-susceptible and -resistant flukes. The altered drug influx/efflux and enhanced metabolic capacity identified in TCBZ-resistant liver flukes may account for the development of resistance to TCBZ.

The mean parasite burden of a population of Tribolium confusum is shown to rise to a plateau as the exposure density of infective eggs of Hymenolepis diminuta increases. The level of this plateau is shown to be dependent on the nutritional status of the host population, being depressed from approximately 18 cysticercoids/beetle in hosts which have been starved prior to experimentation, to approximately 2 cysticercoids/beetle in satiated hosts. A simple model is used to describe the shape of this infection functional response in terms of the predator–prey interaction between hosts (T. confusum) and parasite infective stages (H. diminuta eggs). The distribution of successful infections/host is shown to be over-dispersed, even when hosts are exposed to infective stages arranged in a uniform spatial pattern. The over-dispersion of parasite numbers/host is shown to become more severe as the spatial pattern of infective stages changes from under-dispersed, through random, to over-dispersed. Experimental results are discussed in relation to the dynamics of parasite–host interactions, in which infection takes place by host ingestion of a free-living infective stage.

We have compared the surface radio-iodinated proteins of uninfected and Plasmodium falciparum-infected erythrocytes from natural infections of human patients. Cryopreserved infected blood from Gambian children with falciparum malaria was thawed, cultured to the middle trophozoite stage, and surface radio-iodinated. Trophozoite-infected cells were enriched about 10-fold on a Percoll gradient newly designed to separate cells based on their differential permeability to sorbitol. Infected blood was radio-iodinated and erythrocyes from the fraction enriched in parasitized cells and uninfected erythrocytes from the same sample obtained from the gradient and compared by SDS–PAGE and autoradiography. In each sample, parasitized erythrocytes contained one or more polypeptides of very high molecular weight (Mr 250000–300000) that were not found on uninfected erythrocytes from the same patient. These proteins were isolate-specific in size and number, suggesting that natural isolates contain a variable number of different P. falciparum phenotypes for this surface protein. In addition, these radio-iodinated surface proteins could not be extracted from the host cell membrane by the non-ionic detergent Triton X-100, but were extracted by SDS. The properties of these proteins suggest they are the equivalent for natural infections of the strain-dependent antigen previously described (Leech, Barnwell, Miller & Howard, 1984) on the surface of P. falciparum-infected Aotus erythrocytes. In addition, we observed a second parasite-dependent modification of labelled proteins on infected erythrocytes with the appearance of a new band of Mr 30000. There were also variations in the pattern of radio-isotope labelled proteins on uninfected erythrocytes from different patients.

A large percentage of the cercariae of Schistosoma mansoni die while penetrating the abdominal skin of rats or mice but relatively few cercariae die in the skin of hamsters.

Comparison of hamsters and mice infected from the same pool of cercariae showed that the higher percentage of cercariae recovered as adult worms from hamsters than from mice was due to the lower percentage of cercariae which died during penetration of hamster skin.

In similar comparisons of rats with mice the lower percentage of adult worms recovered from rats was not entirely accounted for by the higher percentage of cercariae which died in rat skin. Losses of schistosomula at later stages of development are apparently higher in rats than in mice.

Cercariae die within 10 min after beginning to penetrate the skin of mice. At this stage the cercariae, which are now termed schistosomula, are attempting to penetrate the Malpighian layer of the epidermis. Five minutes later many schistosomula have entered the dermis.

The cause of death of cercariae has not been determined. It is not related to the development of sensitivity to water which accompanies the transition from cercaria to schistosomulum. Sensitivity to water appears much sooner after penetration in some cercariae than in others; even after 30 min in the skin some cercariae can tolerate a return to water.

In rats, hyper-immunized by a series of infections with cercariae through the abdominal skin, the percentage of challenging cercariae which die in the abdominal skin shows a slight increase over controls. No further mortality of schistosomula occurs during the next 24 h.

Age resistance to infection with S. mansoni could not be demonstrated in comparisons of young mice (1 month) with old mice (1 year) of the Parkes and CBA strain.

We should like to thank Dr F. Hawking for performing the skin biopsies on the dogs and Mrs P. Clark and Miss J. Marsh for their excellent technical assistance.

Monkey blood samples were collected from 214 monkeys relocated as part of the wildlife rescue organized in French Guiana during the filling of the Petit Saut Dam on the Sinnamary River. These samples were tested for malaria parasites by microscopy of thick blood films and by nested PCR for small subunit rRNA genes (SSUrRNA). Parasitic blood forms similar to Plasmodium brasilianum were detected in 4 monkey species: Alouatta seniculus macconnelli, Saguinus midas midas, Pithecia pithecia and Ateles paniscus paniscus, with the highest prevalence in Alouatta monkeys. PCR was more sensitive than the conventional method for detecting low-grade parasitaemia in positive monkeys. The examination of blood films indicated that 5·6% of the animals carried parasites whereas the nested PCR for ribosomal DNA indicated a prevalence of 11·3%. The P. brasilianum SSUrRNA gene sequence was analysed and aligned with those from P. malariae, P. falciparum and P. vivax. This suggested that P. brasilianum and P. malariae are very closely related. Similar results were obtained from analysis of the sequences in P. malariae and P. brasilianum isolates of a polymorphic gene fragment analogous to the merozoite surface protein-1 (MSP-1) gene of P. falciparum. The P. brasilianum/P. malariae sequences were more similar to those of P. vivax than to those of P. falciparum, at least in the gene region examined. The high degree of DNA homology in the sequences of the SSUrRNA and msp1-like genes is consistent with other characterizations demonstrating a taxonomic relationship between P. brasilianum and P. malariae species. Our results provide further evidence that P. brasilianum and P. malariae are virtually identical and should probably be considered to be a single malaria species. This raises the question as to whether monkeys living in the rainforest are natural reservoirs for both simian and human malaria. These results have implications for the interpretation of the current epidemiological situation in French Guiana.

The glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum is thought to function as a critical toxin that contributes to severe malarial pathogenesis by eliciting the production of proinflammatory responses by the innate immune system of mammalian hosts. Analysis of the fine structure of P. falciparum GPI suggests a requirement for the presence of both core glycan and lipid moieties in the recognition and signalling of parasite glycolipids by host immune cells. It has been demonstrated that GPI anchors of various parasitic protozoa can mediate cellular immune responses via members of the Toll-like family of pattern recognition receptors (TLRs). Recent studies indicate that GPI anchors of P. falciparum and other protozoa are preferentially recognized by TLR-2, involving the MyD88-dependent activation of specific signalling pathways that mediate the production of proinflammatory cytokines and nitric oxide from host macrophages in vitro. However, the contribution of malaria GPI toxin to severe disease syndromes and the role of specific TLRs or other pattern recognition receptors in innate immunity in vivo is only just beginning to be characterized. A better understanding of the molecular mechanisms underlying severe malarial pathogenesis may yet lead to substantial new insights with important implications for the development of novel therapeutics for malaria treatment.

The course of the worm burdens of calves infected daily with larvae of Ostertagia ostertagi at five different rates was studied by making post-mortem worm counts on groups of calves killed after experiencing infection for different periods of time.

The relationship between worm numbers and infection rates together with evidence that a turnover of worms occurred, led to the conclusion that burdens of adult worms were regulated primarily by a loss of worms at a rate proportional to the number present.

Further, estimates based on the number of developing fourth-stage larvae indicated that the proportion of the larvae administered which became established decreased gradually as the experiment progressed.

At very low infection rates, the rate of turnover of the worms was slower than at high infection rates but in all groups it remained constant through the 6 months duration of the experiment.

The development of a small proportion of the larvae administered was arrested at the early fourth stage. At the highest infection rate used, the proportion of worms that became inhibited was somewhat greater.

Saponin treatment of cercariae and schistosomula alters the surface membrane so that it may be sheared from the organism and isolated as fragments by centrifugation. Saponin-calcium chloride treatment or freezing and thawing of adult worms removes the surface membrane, which can be washed from the bodies and collected by centrifugation. The small quantities of material available necessitated the development of a sensitive technique for detecting the proteins in the membranes. The surface membranes were radioiodinated after butan-1-ol extraction and electrophoresed in polyacrylamide gel. Adult surfaces prepared by the saponin-calcium chloride and by the freezing and thawing technique had identical protein components, detected as gel-cut profiles or in autoradiographs. The quantity of a rapidly migrating PAS-positive amido black negative component was greater in the surfaces prepared by saponincalcium chloride than in the frozen and thawed surfaces. This component contains lipid, some of which may be glycolipid. It was largely absent from the surface membranes of cercariae and schistosomula. Cercarial surface membranes contained a major protein component which was absent from the surface membranes of schistosomula. Otherwise the surface membranes of the cercariae were identical to those of the schistosomula in their protein components. The rate of incorporation of freshly synthesized protein per unit area of surface membrane of schistosomula was very low in the first 5 days in culture, after which there was a very rapid increase to a maximum rate on the 15th day. After this time, the rate of protein synthesis decreased to a low level at 26 days. In these studies the activity per unit area was measured and this would not be expected to vary greatly during growth. The biological significance of the observed variation in protein incorporation into the membrane is unclear.

Rat peritoneal eosinophils adhere to live Schistosoma mansoni schistosomula in vitro in the presence of fresh normal rat serum, or in heat-inactivated serum from rats immune to the parasite. When the eosinophils are present in sufficient numbers the worms show ultrastructural evidence of surface damage and are ultimately killed. It is believed that the appearance of focal lesions in the tegument of the schistosomulum follows the secretion of enzymes by the eosinophils onto the parasite surface. The cells have been observed within these lesions and later between the basal plasma membrane of the tegument and the underlying interstitial material. It is suggested that the cells are responsible for prising the tegument away from the body of the worm. The detached tegument shows evidence of further degradation. Adherent eosinophils which have released their secretions appear to degenerate and are eventually replaced by macrophages. Remnants of both the expended eosinophils and the disrupted tegument have been identified within the macrophages. Adherence of eosinophils through C3–C3 receptor interaction results in earlier and more severe damage to the schistosomula than when adherence occurs through Fe receptors. Rat eosinophils also adhere to C3-coated, glutaraldehyde-flxed schistosomula and C3-coated Sepharose beads. However, evidence of enzyme secretion is only obtained when the target is a schistosomulum.

Observed field data from a range of geographically distinct human communities suggest a consistent non-linear relationship between prevalence and mean intensity of Ascaris lumbricoides infection. Utilizing the negative binomial distribution as a description of observed aggregation, maximum-likelihood analysis reveals that the degree of aggregation is a negative linear function of mean worm burden. The factors responsible for this relationship in human populations require further study but may involve some combination of (i) density-dependent reduction in worm numbers within individuals, (ii) density-dependent parasite-induced host mortality or (iii) self-treatment by heavily infected hosts. Variability in the degree of aggregation appears dependent on the level of infection in a community and independent of geographical differences in the host or parasite populations.

Neospora caninum and Toxoplasma gondii are closely related intracellular protozoan parasites associated with bovine and ovine abortion respectively. Little is known about the extent of Neospora/Toxoplasma co-infection in naturally infected populations of animals. Using nested PCR techniques, based on primers from the Nc5 region of N. caninum and SAG1 for T. gondii, the prevalence of N. caninum and its co-infection with T. gondii were investigated in populations of Mus domesticus, Rattus norvegicus and aborted lambs (Ovis aries). A low frequency of infection with N. caninum was detected in the Mus domesticus (3%) and Rattus norvegicus (4·4%) populations. A relatively high frequency of infection with N. caninum was detected in the brains of aborted lambs (18·9%). There was no significant relationship between N. caninum and T. gondii co-infection. Investigation of the tissue distribution of Neospora, in aborted lambs, showed that Neospora could not be detected in tissues other than brain and this was in contrast to Toxoplasma where the parasite could be frequently detected in a range of tissues.

Toxoplasma gondii is a highly ubiquitous and prevalent parasite. Despite the cat being the only definitive host, it is found in almost all geographical areas and warm blooded animals. Three routes of transmission are recognised: ingestion of oocysts shed by the cat, carnivory and congenital transmission. In natural populations, it is difficult to establish the relative importance of these routes. This paper reviews recent work in our laboratory which suggests that congenital transmission may be much more important than previously thought. Using PCR detection of the parasite, studies in sheep show that congenital transmission may occur in as many as 66% of pregnancies. Furthermore, in families of sheep on the same farm, exposed to the same sources of oocysts, significant divergent prevalences of Toxoplasma infection and abortion are found between different families. The data suggest that breeding from infected ewes increases the risk of subsequent abortion and infection in lambs. Congenital transmission rates in a natural population of mice were found to be 75%. Interestingly, congenital transmission rates in humans were measured at 19·8%. The results presented in these studies differ from those of other published studies and suggest that vertical transmission may be much more important than previously thought.

Hookworms are gut-dwelling, blood-feeding nematodes that infect hundreds of millions of people, particularly in the tropics. As part of a program aiming to define novel drug targets and vaccine candidates for human parasitic nematodes, genes expressed in adults of the human hookworm Necator americanus were surveyed by the expressed sequence tag approach. In total 161 new hookworm genes were identified. For the majority of these, a function could be assigned by homology. The dataset includes proteases, protease inhibitors, a lipid binding protein, C-type lectins, an anti-bacterial factor, globins and other genes of interest from a drug or vaccine development viewpoint. Three different classes of small, secreted proteins were identified that may be involved in the host–parasite interaction, including potential potassium channel blocking peptides. One third of the genes were novel. These included highly expressed, secreted (glyco)proteins which may be part of the excretory–secretory products of these important pathogens. Of particular interest are a family of 9 genes with similarity to the immunomodulatory protein, neutrophil inhibitory factor, that may play a role in establishing an immunocompromised niche for this successful parasite.

1. The following new species were encountered in this investigation:

Haemogregarina hamata in Lacerta jacksoni;

Hepatozoon minchini in Crotaphopeltis degeni

Pirhaemocyton granosa in Agama colonorum

Atoxoplasma argyae n.gen. n.sp. in Argya rubi-ginosa

Spirochaeta harveyi in Cercopithecus aethiops.

2. Further information is given in regard to the following known species, including new host records:

Plasmodium pitmani in Mabuia striata and M. maculilabris

Trypanosoma martini in Mabuia maculilabris

Plasmodium vaughani in Othyphantes reichenowi

Plasmodium relictum in Othyphantes reichenowi and Passer iagoensis

Haemoproteus wenyoni in Passer griseus

Leucocytozoon sp. in nine species of birds

Hepatocystes epomophori in Epomophorus labiatus

Hepatocystes kochi in Cercopithecus, etc.

Babesia pitheci in Cercopithecus mitis.

3. Exo-erythrocytic schizogony is described in Plasmodium pitmani of the skink (from spleen and heart preparations).

Malaria is a disease that still affects a significant proportion of the global human population. Whilst advances have been made in lowering the numbers of cases and deaths, it is clear that a strategy based solely on disease control year on year, without reducing transmission and ultimately eradicating the parasite, is unsustainable. This article highlights the current mainstay treatments alongside a selection of emerging new clinical molecules from the portfolio of Medicines for Malaria Venture (MMV) and our partners. In each case, the key highlights from each research phase are described to demonstrate how these new potential medicines were discovered. Given the increased focus of the community on eradicating the disease, the strategy for next generation combination medicines that will provide such potential is explained.

Host responses to the transmittable stages of the malaria parasite may reduce transmission effectively. Transmission-reducing activity (TRA) of human serum can be determined as a percentage, using the Standard Membrane Feeding Assay (SMFA). This laboratory assay was evaluated using the results of 121 experiments with malaria-endemic sera among which many repeated measurements were obtained. The assay consists of the feeding of Anopheles stephensi mosquitoes with cultured Plasmodium falciparum gametocytes, mixed with human red blood cells, and control and experimental sera. The TRA of individual sera was determined by the comparison of oocyst densities between these sera. Bootstrap data on oocyst densities in individual mosquitoes in control feeds were used to construct confidence limits for TRA percentages of serum feeds. Low (<20%) and high TRA (>90%) values for individual sera were usually reproduced in a second experiment, whereas this was more difficult for values between 20% and 90%. The observed variability of TRA values is explained in part by the variability in oocyst density per mosquito. Oocyst densities in control feeds varied more between experiments than within experiments and showed a slight decline over the 3 years of experiments. Reproducibility of TRA of field sera was low (20%) between experiments, but much higher (61%) within experiments. A minimum of 35 oocysts per mosquito in control feeds gave optimal reproducibility (44%) between experiments. We recommend that (1) sera are compared within an experiment, or (2) assays are only analysed where controls have at least 35 oocysts per mosquito. The SMFA is under the recommended conditions appropriate for the study of factors that may influence TRA, e.g. transmission blocking vaccines.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a ‘proteinase-enriched’ Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT–PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

The northern biotype of Echinococcus granulosus occurs throughout the holarctic zones of tundra and taiga, from eastern Fennoscandia to the Bering Strait in Eurasia and in North America from arctic Alaska approximately to the northern border of the United States. The cycle of the cestode is complex in taiga at lower latitudes, because of the greater diversity of potential hosts. In the Arctic and Subarctic, however, four patterns of predator/prey relationships may be discerned. Two natural cycles involve the wolf and wild reindeer and the wolf and elk (moose), respectively. Where deer of the two species coexist, both are prey of the wolf; the interactions of the wolf and elk are here described on the basis of long-term observations made on Isle Royale (in Lake Superior near the southern limit of taiga), where only the wolf and elk serve as hosts for E. granulosus. A synanthropic cycle involving herding-dogs and domesticated reindeer caused hyperendemicity of cystic echinococcosis in arctic Eurasia, mainly in northeastern Siberia. The 4th pattern, a semi-synanthropic cycle, formerly existed in Alaska, wherein sled-dogs of the indigenous hunters became infected by consuming the lungs of wild reindeer. The sequence of changes in life-style inherent in the process of acculturation affected the occurrence of cystic echinococcosis among nomadic Iñupiat in arctic Alaska. When those people became sedentary, the environs of their early villages soon became severely contaminated by faeces of dogs, and cases of cystic echinococcosis occurred. Compared to cystic echinococcosis caused by E. granulosus adapted to synanthropic hosts (dog and domestic ungulates), the infection produced by the northern biotype is relatively benign. Nearly all diagnosed cases of cystic echinococcosis (>300) in Alaska have occurred in indigenous people; only one fatality has been recorded (in a non-indigenous person). After sled-dogs were replaced by machines, cases have become rare in Alaska. A similar effect has been observed in Fennoscandia, in the Saami and domesticated reindeer. Recent records indicate that the prevalence of cystic echinococcosis is increasing in Russia, suggesting that dogs are used there in herding.

Genomes of the major human helminth parasites, and indeed many others of agricultural significance, are now the research focus of intensive genome sequencing and annotation. A draft genome sequence of the filarial parasite Brugia malayi was reported in 2007 and draft genomes of two of the human schistosomes, Schistosoma japonicum and S. mansoni reported in 2009. These genome data provide the basis for a comprehensive understanding of the molecular mechanisms involved in schistosome nutrition and metabolism, host-dependent development and maturation, immune evasion and invertebrate evolution. In addition, new potential vaccine candidates and drug targets will likely be predicted. However, testing these predictions is often not straightforward with schistosomes because of the difficulty and expense in maintenance of the developmental cycle. To facilitate this goal, several developmental stages can be maintained in vitro for shorter or longer intervals of time, and these are amenable to manipulation. Our research interests focus on experimental studies of schistosome gene functions, and more recently have focused on development of transgenesis and RNA interference with the longer term aim of heritable gene manipulation. Here we review methods to isolate and culture developmental stages of Schistosoma mansoni, including eggs, sporocysts, schistosomules and adults, in particular as these procedures relate to approaches for gene manipulation. We also discuss recent advances in genetic manipulation of schistosomes including the deployment of square wave electroporation to introduce reporter genes into cultured schistosomes.

Solid experimental evidence indicates that EBA-175 is used as a ligand by the Plasmodium falciparum merozoite to bind to human RBC, via different binding processing fragments. Using synthetic peptides and specific receptor-ligand interaction methodology, we have identified 6 high-activity binding sequences from the EBA-175 CAMP strain; peptide 1758 (KSYGTPDNIDKNMSLIHKHN), located in the so-called region I for which no binding activity has been reported before, peptides 1779 (NIDRIYDKNLLMIKEHILAI) and 1783 (HRNKKNDKLYRDEWWKVIKK), located in region II, in a sub-region known as 5′ Cys F2, previously reported as being a binding region, and peptides 1814 (DRNSNTLHLKDYRNEENERH), 1815 (YTNQNINISQERDLQKHGFH) and 1818 (NNNFNNIPSRYNLYDKKLDL), in region III–V where antibodies inhibit merozoite invasion of erythrocytes. The affinity constants were between 60 and 180 nM and the critical amino acids involved in the binding were identified. The binding of these peptides to enzyme-treated RBC was analysed; binding of peptide 1814, located in the III–V region, was found to be sialic acid dependent. Some of these high binding peptides were able to inhibit in vitro merozoite invasion and to block the binding of recombinant RII-EBA to RBC. Several of these peptides are located in regions recognized by protective immune clusters of merozoites (ICMs) eluted antibodies.