Two full-length beta-tubulin cDNAs, representing isotypes 1 and 2, were cloned from the cattle nematode Cooperia oncophora. The predicted protein sequences span 448 amino acids, and show a high degree of identity to beta-tubulins from other nematodes. While C. oncophora isotype 1 sequence had the highest identity to Haemonchus contortus isotype 1 and Teladorsagia circumcincta sequences (95% identity), the C. oncophora isotype 2 sequence was most similar to H. contortus isotype 2 and Trichostrongylus colubriformis (92% identity). Alignment of the two C. oncophora sequences with other trichostrongylid beta-tubulins deposited in GenBank showed a clear distinction between isotype 1 and 2 beta-tubulin classes. The two classes differed at 19 amino acid positions, most notably at the carboxy terminus. These isotype-defining residues were conserved among different trichostrongylid species within a class. Analysis of fragments of both genes revealed a high degree of genetic variability in coding and non-coding regions. However, all nucleotide differences detected in the coding region were silent, as they did not result in any amino acid substitution. Analysis of 2 groups of worms for the codon 200 polymorphism associated with benzimidazole resistance revealed a proportion of worms in 1 of the groups bearing a tyrosine at this position.

The rapid elimination of the intestinal phase of Trichinella spiralis in NIH mice is associated with progressive inflammation of the intestinal tract. The non-specific effects of this inflammation were studied in. mice concurrently infected with an unrelated parasite, Hymenolepis diminuta, which does not stimulate a visible inflammatory response but is also immunologically rejected by this strain of mice.

It was demonstrated that the rejection phase of T. spiralis infection had a marked effect upon the growth and survival of H. diminuta,. The cestode either failed to establish or to grow; if the worms were already strobilate when inflammation developed then destrobilation occurred. There was no cross–immunity between the parasites, nor was the interaction a direct consequence of inter–specific competition.

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4–5·5, 37 °C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without efifect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial eflfect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

1. The anatomy of the adult miracidium of Parorchis acanthus is described.

2. Development of the miracidium and of the succeeding larval stages has been followed.

3. Cleavage of the fertilized egg gives rise to an ectodermal and a propagatory cell. The former gives rise to the soma, and the latter, after contributing a few small cells to the body of the miracidium, becomes the germ cell.

4. This germ cell develops in the same way into the first generation redia while still in the body of the miracidium.

5. The fully formed miracidium contains a single fully formed redia when it penetrates the intermediate molluscan host.

6. In the intermediate host the miracidium disintegrates and second generation rediae are formed from the germ cells in the body cavity of the parent redia.

7. The germ cells in the body cavity of the daughter redia give rise to cercariae by a similar process of embryonic development to that found in preceding generations.

8. The germ cells in the cercariae become differentiated into the adult genitalia.

9. No evidence has been found to show that rediae or cercariae are derived from cells in the wall of the redia.

10. No ovary, maturation process, parthenogenesis, or fertilization takes place in any of the larval stages, but germinal lineage is apparent throughout the life cycle.

A primary objective of schistosomiasis control programmes is to achieve, and hence also demonstrate, a quantifiable reduction in schistosome-associated morbidity as a consequence of chemotherapeutic intervention. Inherent within such an objective, it is necessary to define and validate direct and indirect indicators of schistosome-related morbidity. However, to define and thereby document such morbidity, and its reduction following treatment, may not be straightforward, particularly for intestinal schistosomiasis-induced morbidity, which is often not apparent in all but the most severe or chronic cases. Within all ‘Schistosomiasis Control Initiative’ activities, across selected sub-Saharan African countries since 2002, a range of standard and novel potential morbidity markers have been monitored and evaluated. Parasitological intensity measures, combined with haemoglobin/anaemia counts and ultrasonography, proved valuable schistosomiasis-related morbidity indicators, being both logistically practical and informative. Additional measures tested, such as albumin excretion profiles, were promising, and are subject to ongoing research, whilst some measures, such as distended stomach/umbilical circumference, anthropometrics and health questionnaires proved less reliable. These results serve to both illustrate the success of current control activities in reducing schistosome-induced morbidity, and to highlight key tools and techniques for continued application within ongoing and future mass drug administration programmes.

Investigations were carried out on the influence of the local environment on the preparasitic stages of B. phlebotomum and the following observations were made.

1. The infective larvae are positively thermo-tropic and positively phototropic. These observations confirm those of Schwartz (1924). The larvae are not negatively geotropic and do not climb grass, but remain in the dung, gaining access to the host by adherence of the dung to the skin.

2. Lack of air, such as would occur in tightly packed faeces; immersion of the faeces in water; temperatures below 10° C.; and lack of moisture, are all inhibitory to development. Infective larvae are resistant to at least 5 days‘ direct exposure to an atmosphere of relative humidity 75 at a temperature of 25° C.; in dry faeces they are resistant to 14 days‘ exposure to the same atmosphere.

3. In Northern Nigeria desiccation is the most important inhibitory factor in development. The extreme dryness of the pastures in the dry season prevents development of the larvae and adherence of dung to the skin. Penetration of the skin of the host probably occurs only in the rainy season, maximal penetration probably occurring just after the rains are regularly established.

4. The female lays 600 eggs in 12 hr. in the early rains, but the output of eggs by the females varies considerably throughout the year. It is greatest in the early rains and lowest in the later months of the dry season.

5. The female hookworm burden of nomadic cattle fluctuates throughout the year. The maximum burden occurs in the later months of the dry season.

6. Three factors, all probably bound up with a single factor, i.e. moisture, seem to influence the degree of pasture infectivity. (a) Inhibition of larval development; (b) failure of faeces to adhere to the skin; (c) fluctuation in the egg-laying rate of the female hookworms. The fluctuating pasture infectivity results in a fluctuating hookworm burden.

7. The period of maximum hookworm burden in nomadic cattle more or less coincides with the period of incidence of a disease in Stock Farm cattle, associated with hookworm infestation.

In the discussion an attempt is made to correlate these observations both in accounting for the seasonal incidence of ‘hookworm disease’ and in suggesting methods of control.

This paper was written during the tenure of a Cooper Centenary Fellowship for which the author is indebted to the Council of the Veterinary Educational Trust. The writer's thanks are also due to his laboratory assistant in Nigeria, Mr R. A. O. Shonekan.

In an attempt to determine the mechanisms of resistance to schistosome infection operative in bulinid snails, an histological study was carried out on batches of a refractory strain of Bulinus truncatus and two highly susceptible strains of B. africanus killed at various intervals of time, ranging from 7 h to over 3 months, after exposure to infection with Schistosoma mattheei miracidia.

It was found that only a small fraction (less than 1%) of the miracidia penetrated B. truncatus, and that they penetrated only a few of the snails exposed. Much larger proportions (up to 26%) of the miracidia penetrated B. africanus and they penetrated nearly all the snails exposed.

In the susceptible B. africanus only miracidia which settled in the loose tissues of the head-foot proceeded to develop: those settling in dense tissue failed to develop and degenerated within about 2 days.

A strong infiltration reaction of amoebocytes rapidly destroyed the few miracidia that successfully penetrated B. truncatus. In contrast, B. africanus did not respond to the presence of miracidia in its tissues, nor to mother or to daughter sporocysts before the onset of cercarial shedding: it reacted only to cercariae.

It is concluded (a) that the combination of an effective surface barrier and an active defence mechanism due to amoebocytes were responsible for complete resistance to S. mattheei infection in B. truncatus (b) that successful establishment of S. mattheei infection in B. africanus depended on the failure of the snail to recognize the schistosome miracidia and sporocysts as foreign material.

I am indebted to Professor George S. Nelson for advice and encouragement in this work which was carried out at the London School of Hygiene and Tropical Medicine. I am most grateful to Dr R. J. Pitchford, Dr C. A. Wright and Dr F. Arfaa for providing material for schistosome and snail cultures and to the East African Community and the British Ministry of Overseas Development for financial support.

This review describes some of the developments in helminth biochemistry that have taken place over the last 40 years. Since the early 1970s the main anabolic and catabolic pathways in parasitic helminths have been worked out. The mode of action of the majority of anthelmintics is now known, but in many cases the mechanisms of resistance remain elusive. Developments in helminth biochemistry have depended heavily on developments in other areas. High throughput methods such as proteomics, transcriptomics and genome sequencing are now generating vast amounts of new data. The challenge for the future is to interpret and understand the biological relevance of this new information.

1. The osmotic pressure of the medium in which the nematodes were cultured has been shown to be equivalent to that of about 30 mM. NaCl.

2. The effects of immersion in a saline solution isotonic with the above, in glass-distilled water, and in hypertonic saline solutions, have been described. Particular attention was paid to changes in the length of the body, in the appearance and behaviour of the alimentary canal, and in the movement of the body.

3. The results obtained have been discussed, and the following tentative conclusions reached:

(a) The main aqueous exchanges occur through the cuticle and protoplasm covering the periphery of the body, and are controlled by the protoplasm rather than by the cuticle. The cuticle thus does not act as aja impermeable outer covering which insulates the animal from its environment.

(b) The species normally lives in an environment which is hypotonic to the body fluids, and maintains its internal osmotic pressure by means of an active method of osmotic regulation. The alimentary canal is probably partially, but not wholly, responsible for this regulation.

(c) Swelling occurs in distilled water because the removal of water in the initial stages is not rapid enough to cope with the extra inflow. Later the excess water is removed.

(d) In concentrated solutions, water is removed from the body, and the internal osmotic pressure may also be increased to some extent by the penetration of osmotically active substances to the body, through the cuticle and hypodermis of the body wall. Penetration through the hypodermis is slower than through the cuticle.

(e) During recovery in concentrated solutions, there is a slow removal of osmotically active material from the body. The hypertrophy of the lateral and post-anal organs, which sometimes occurs under these conditions, may be connected with this removal.

Gyrodactylus specimens infecting both anadromous Arctic charr (Salvelinus alpinus) from River Signaldalselva (northern Norway) and resident Arctic charr from Lake Pålsbufjorden (southern Norway) were identified as G. salaris using molecular markers and morphometrics. The infection in Pålsbufjorden represents the first record of a viable G. salaris population infecting a host in the wild in the absence of salmon (Salmo salar). G. salaris on charr from Signaldalselva and Pålsbufjorden bear different mitochondrial haplotypes. While parasites infecting charr in Signaldalselva carry the same mitochondrial haplotype as parasites from sympatric Atlantic salmon, G. salaris from charr in Pålsbufjorden bear a haplotype that has previously been found in parasites infecting rainbow trout (Oncorhynchus mykiss) and Atlantic salmon, and an IGS repeat arrangement that is very similar to those observed earlier in parasites infecting rainbow trout. Accordingly, the infection may result from 2 subsequent host-switches (from salmon via rainbow trout to charr). Morphometric analyses revealed significant differences between G. salaris infecting charr in the 2 localities, and between those on sympatric charr and salmon within Signaldalselva. These differences may reflect adaptations to a new host species, different environmental conditions, and/or inherited differences between the G. salaris strains. The discovery of G. salaris on populations of both anadromous and resident charr may have severe implications for Atlantic salmon stock-management as charr may represent a reservoir for infection of salmon.

The metacyclic form of African trypanosomes is the first to express genes for the Variant Surface Glycoprotein (VSG) and it uses an unusually predictable subset of the VSG gene repertoire. We have developed a model system for the analysis of metacyclic VSG (M-VSG) gene expression and have used this to demonstrate that, for two M-VSG genes, different modes of expression operate in the insect and mammalian phases of the life-cycle. In metacyclic-derived clones, these genes are expressed in situ, whereas they are routinely activated by duplication in bloodstream trypanosomes. The expression loci for both M-VSG genes studied are structurally simple and we present a model, based on this, for the maintenance of a separate M-VSG repertoire and expression system.

The tegument of Proteocephalus pollanicoli is organized at two levels. The external level is a syncytial layer of protoplasm composed of vesicles in a granular matrix. The surface of this level has microvillus-like structure, microtriches, with sharp electron dense tips and less dense bases. Pinocytotic vesicles, large vacuoles, and evaginations occur at the surface of the external level, whereas mitochondria and lipid droplets tend to be concentrated basally. The basal plasma membrane has in some places small invaginations and at others large evaginations forming protoplasmic tubes which connect the external and internal levels.

The internal level is composed of individual areas of dense, granular protoplasm, each of which has a nucleus. These areas, or cuticular cells, contain lipid droplets, mitochondria, short lengths of agranular endoplasmic reticulum and phospholipid bodies. Two cuticular cells lying close together may be joined by a protoplasmic bridge and thereby form a limited syncytium.

The morphology of the tegument of this proteocephalid is compared with that of the cyclophyllidean species and the function of the tegument in parasitic helminths is discussed.

I should like to acknowledge the technical assistance of Mr W. Ferguson and Mr A. Lyness. My thanks are also due to the Wellcome Trust for a grant to purchase the Akashi Transcope and ancillary equipment.

Within aquatic habitats, the hyper-abundant Order Crustacea appear to be the predominant host group for members of the Phylum Microsporidia. The musculature, a common site of infection, provides access to biochemical (carbohydrate-rich) and physiological (mitochondria-rich) conditions conducive to prolific parasite replication and maturation. The significant proportion of body plan devoted to skeletal musculature in Crustacea provides the location for a highly efficient intracellular parasite factory. In this study, we utilize histological, ultrastructural and phylogenetic evidence to describe a previously known (Inodosporus octospora) and novel (Ovipleistophora arlo n. sp.) microsporidian parasites infecting the musculature of the common prawn (Palaemon serratus) from the same site, at the same time of year. Despite similar clinical signs of infection, both parasites are otherwise distinct in terms of pathogenesis, morphology and phylogeny. Based upon partial subunit ribosomal RNA (SSU rDNA) sequence, we show that that I. octospora may be identical to a Kabatana sp. previously described infecting two-spot goby (Gobiusculus flavescens) in Europe, or at least that Inodosporus and Kabatana genera are synonyms. In addition, SSU rDNA sequence for O. arlo places it within a distinct clade containing Ovipleistophora mirandellae and Ovipleistophora ovariae, both infecting the oocytes of freshwater fish in Europe. Taken together, our data provide strong evidence for trophic-transfer between crustacean and fish hosts for two different microsporidians within clade 5 of the phylum. Furthermore, it demonstrates that morphologically and phylogenetically distinct microsporidians can infect the same tissues of the same host species to impart clinical signs which mimic infection with the other.

A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5·0. The optimal pH and temperature of the enzyme were 7·5 and 28 °C respectively, using α-N-ρ-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 °C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin β-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E13-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized.

Parasites can exert strong effects on population to ecosystem level processes, but data on parasites are limited for many global regions, especially tropical marine systems. Characterizing parasite diversity and distributions are the first steps towards understanding the potential impacts of parasites. The Panama Canal serves as an interesting location to examine tropical parasite diversity and distribution, as it is a conduit between two oceans and a hub for international trade. We examined metazoan and protistan parasites associated with ten oyster species collected from both Panamanian coasts, including the Panama Canal and Bocas del Toro. We found multiple metazoan taxa (pea crabs, Stylochus spp., Urastoma cyrinae). Our molecular screening for protistan parasites detected four species of Perkinsus (Perkinsus marinus, Perkinsus chesapeaki, Perkinsus olseni, Perkinsus beihaiensis) and several haplosporidians, including two genera (Minchinia, Haplosporidium). Species richness was higher for the protistan parasites than for the metazoans, with haplosporidian richness being higher than Perkinsus richness. Perkinsus species were the most frequently detected and most geographically widespread among parasite groups. Parasite richness and overlap differed between regions, locations and oyster hosts. These results have important implications for tropical parasite richness and the dispersal of parasites due to shipping associated with the Panama Canal.

The antimalarial drug mefloquine has promising antischistosomal properties killing haematophagous adult schistosomes as well as schistosomula. The mode of action and involved drug targets of mefloquine in Schistosoma mansoni schistosomula are unknown. In order to identify mefloquine-binding proteins and thus potential drug targets, mefloquine affinity chromatography with S. mansoni schistosomula crude extracts was performed. We found one specific mefloquine-binding protein that was identified by mass spectrometry as the glycolytic enzyme enolase (Q27877). Enolase activity assays were performed on schistosomula crude extracts and on the recombinant enolase Q27877 expressed in Escherichia coli. In schistosomula crude extracts enolase activity was inhibited by mefloquine and by the enolase inhibitor sodium fluoride, while activity of the recombinant enolase was not affected. In contrast to enolase from crude extracts, recombinant Q27877 did not bind to mefloquine-agarose. Using isothermal microcalorimetry, we next investigated the metabolic inhibition of mefloquine and 3 known glycolytic inhibitors in Schistosoma spp., namely sodium fluoride, 3-bromopyruvate and menadione on schistosomula in the presence or absence of glucose. We found that in the presence of glucose, schistosomula were less affected by mefloquine, sodium fluoride and 3-bromopyruvate, whereas glucose had no protective effect when schistosomula had been exposed to menadione. These results suggest a potential role of mefloquine as an inhibitor of glycolysis, at least in stages where other targets like haem degradation are not relevant.

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

The immune status of a host infected with Trypanosoma spp. or Leishmania spp. can play an important role in successful chemotherapy. In animal models, treatment of African trypanosomiasis with difluoromethylornithine or melarsoprol requires an appropriate antibody-mediated immune response. An intact immune system is also necessary for rapid clearance of trypanosomes from the bloodstream following treatment with suramin or quinapyramine. Similarly, an efficient cell-mediated immune response is required for maximal efficacy of pentavalent antimonials in the treatment of leishmaniasis. However, the potential relationship between parasite-induced or acquired immunosuppression and effective chemotherapy has been poorly studied. Macrophages which have been activated by bacterial cell wall components or gamma-interferon are known to display increased activity against Leishmania donovani or Trypanosoma cruzi. In experimental and clinical visceral leishmaniasis, use of macrophage activators together with pentavalent antimonials has lowered the dose of antimony required to cure the infection.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

During the acute phase of Trypanosoma cruzi infection, strong haematological and immune system alterations are observed. The parasite expresses trans-sialidase, a virulence factor responsible for the sialylation of its surface glycoconjugates. This enzyme is also shed to the bloodstream where it is associated with immune system alterations triggered during the infection. During experimental and human infections, the host elicits antibodies able to neutralize the enzyme activity that would be responsible for restricting systemic trans-sialidase to the early steps of the infection, when major immune alterations are induced. The actual relevance of these antibodies was tested by passive transference of monoclonal neutralizing antibodies in acute infection models displaying extreme sensitivity to the infection. Mice were inoculated with virulent parasite strains that induce high parasitaemia, early mortality and strong immune tissue abnormalities. The trans-sialidase-neutralizing antibodies were able to preserve B cell areas both in ganglia and spleen as well as the thymus architecture even in these extreme models. Although no differences between control and treated mice regarding animal survival were found, a major role for the humoral response in controlling the damage of the immune system induced by a systemically distributed virulence factor was defined in an infection with a eukaryotic pathogen.