Toxoplasmosis is a worldwide zoonosis. However, data from Portugal are limited and a considerable part of the literature is in Portuguese. Currently, the rate of congenital infection in Portugal is unknown, and almost nothing is known of sequelae of congenital toxoplasmosis. There is no recent general population-based serological survey of Toxoplasma gondii in humans in Portugal. In addition, there is little information on genetic characteristics of T. gondii in animals and humans. In the present paper, we review prevalence, clinical spectrum and epidemiology of T. gondii in humans and animals in Portugal. This knowledge should be useful to biologists, public health workers, physicians and veterinarians.

The cloning and molecular characterization of a cDNA encoding Ascaris suum 24 kDa antigen (As24) are described. The cDNA sequence consists of 853 bp with an open reading frame coding for a protein of 147 amino acids with an inferred signal peptide of 19 amino acids. The predicted molecular mass and pI were 16 kDa and 8·35 respectively. The endogenous protein in adult A. suum was 24 kDa with the expected pI. A search of the public databases revealed over 50% homology with proteins from filarial parasites but not to other known proteins, suggesting that As24 is a nematode-specific protein. Immunohistochemical studies using polyclonal antibodies raised against Escherichia coli-expressed recombinant As24 demonstrated that the endogenous As24 proteins were intensely localized in unembryonated eggs within the uterus, uterine and gut epithelium, muscle tissues and in the hypodermis of an adult female A. suum. Endogenous As24 was expressed throughout A. suum development and was detected in the excretory/secretory products by immunoblot analysis. Importantly, a homologous protein(s) was detected in Ascaris from human and Toxocara canis from dog, suggesting that As24 is a nematode-specific protein.

Previous research has indicated that supplementing an apparently adequate diet with additional protein improves both host resistance and resilience in lambs infected with Haemonchus contortus. The present study tested the influence of supplementation with non-protein nitrogen (urea). Helminth-naive Hampshire Down lambs were given an apparently adequate basal diet or a diet supplemented with urea. The lambs were then infected with Haemonchus contortus for 10 weeks. Supplementation with urea had no discernible effect on resistance to infection; faecal egg counts, worm burdens, worm lengths and mean number of eggs per adult female worm did not differ between the 2 groups. However, lambs on the supplemented diet showed better resilience; they had greater packed red cell volumes, higher plasma albumin concentrations and increased liveweight gain compared to lambs on the basal diet. The loss of appetite following infection was less in lambs fed the urea-supplemented diet. The observed effect of urea supplementation was seemingly due to greater food consumption as well as the better diet.

When bovine peripheral blood leucocytes were infected with Theileria parva sporozoites, immediate treatment with oxytetracycline (OTC) inhibited the development of sporozoites to mature schizonts. The extent of inhibition was dependent on drug concentration and duration of treatment. Concentrations of 5 μg/ml OTC, or higher, for 8 days completely inhibited the establishment of schizonts and their ability to transform host cells. A cytostatic effect on schizont-infected cell lines was found with three tetracyclines and was also demonstrated on uninfected lymphoblasts. The parasites were found to be sensitive t OTC during development to schizonts, but when mature and established within host cells, schizonts were not demonstrably affected. The infectivity of sporozoites and the binding of sporozoites to lymphocytes were not directly inhibited by OTC. The results may explain the action of tetracyclines when used prophylactically during immunization against East Coast fever, and also the reasons for the ineffectiveness of these drugs when used therapeutically during patent disease.

An improved perfusion apparatus for the cultivation of malaria parasites is described. It consists of a sandwich of three chambers, of which the outer two contained medium and the inner contained the infected blood sample.

Using this apparatus and rocker-dilution cultures it has been shown that medium ‘199’ and medium ‘NCTC 135’ are as good as the Harvard medium for the cultivation of P. knowlesi in vitro.

The asexual cycle of P. knowlesi took slightly longer in vitro than in vivo. The stage of development at which the parasite was inoculated into culture affected the amount of reinvasion of new host cells. Thus, a greater amount of re-invasion was obtained with an inoculum of schizonts than with one of ring stage parasites. The rate of multiplication of the parasite decreased at each successive subculture.

I should like to thank Dr F. Hawking and Dr J. Williamson for their advice, Mr T. J. Scott-Finnigan for technical assistance, Mr F. R. Wanless and Mr C. D. Sutton for taking the photographs, and Mr F. A. New for drawing the figures. The perfusion apparatus was made by Mr T. Harding of the Engineering Division, National Institute for Medical Research. This work received financial assistance from the World Health Organization.

1. Methods of sampling pastures for infective larvae are discussed.

2. An account is given of the methods of operation and identification of larvae from herbage.

3. The distribution of larvae on pastures is discussed in relation to choice of method of sampling and interpretation of results.

4. The significance of grazing patterns to distribution of larvae is considered.

5. An attempt is made to evaluate the use and limitations of pasture sampling methods.

The author is indebted to the Agricultural Research Council for financial assistance in the form of a special research grant.

Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

Observations are described on Polymorphus minutus in its final and intermediate hosts from a wildfowl reserve in Kent. The appearance of the parasite in the reserve followed the alteration of the course of the River Darent, an operation which created a suitable habitat for the intermediate host, Gammarus pulex.

The parasite has been found to occupy the same region of the intestine in the Mallard as in domestic ducks, but it occupies a more anterior region in the Tufted Duck. Worms of varying age have been shown to be present in natural infections of wildfowl.

P. minutus and a cestode, Dicranotaenia coronula, appear to compete for attachment sites in the intestine of Mallard. This competition may be a factor which causes the attachment zone of P. minutus to be extended anteriorly.

The presence of the parasite in the reserve appears to be causing the population of G. pulex to decrease.

It is suggested from the observations made on the birds of the reserve, from the results of infecting domestic ducks and from a consideration of the list of final hosts of the parasite, that there exist strains of P. minutus, adapted to development in different species of birds.

We wish to thank Dr P. Tate for helpful discussions, and Dr I. C. Williams, Department of Zoology, University of Hull, for the identification of the cestodes and criticism of the manuscript. Thanks are also due to Drs J. M. and D. L. Harrison, Mr G. Wallis and Mr A. Meikle for assistance with the population studies and the collection of some of the material.

In this paper we describe the effect of poly-L-lysines of different molecular weight on the schistosomula. In the control sample, the schistosomula of Schistosoma mansoni take up fluorescent Texas Red conjugated to bovine serum albumin (TxR–BSA) into the gut. Following slight damage by 24·0 kDa poly-L-lysine, a high proportion of schistosomula take up fluorescent TxR–BSA into the excretory system. Subsequently, the dye diffused into the bodies of the schistosomula. We suspected that this diffusion involved the process of endocytosis so we investigated this with the use of endocytosis inhibitor, Latrunculin A. Addition of the endocytosis inhibitor Latrunculin A following poly-L-lysine treatment inhibited gut uptake of TxR–BSA as well as the diffusion of excretory-ingested TxR–BSA molecules.

In the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts of Spirometra mansoni plerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE–anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7·5 and 0·1 M Tris–HCI. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors, and 4-(amidinophenyl)methansulfonyl fluoride (APMSF, 0·5 m) and N-α–p–tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK, I mM) and chymostatin (0·1 mM), and was potentiated in 10 mM Ca2+ which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff(PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-γ (rIFNγ) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.

(1) Herpetomonas jaculum is a parasite of Nepa cinerea, occurring in the alimentary tract of its host.

(2) The life-history of the parasite may be conveniently divided into three stages, the pre-flagellate, flagellate and post-flagellate stages, which gradually merge into one another.

(3) The movements of the parasite are less flexible than those of Crithidia, as Herpetomonads have no undulating membrane. The flagellum is the most active agent in effecting motion.

(4) The pre-flagellate stages of the parasite (Pl V, Figs. 1–18) are best observed in the crops of nymphs of Nepa cinerea. The parasites at first are oval (Figs. 1–6). They vary in size from about 4μ to 5μ long and from 2μ to 2·5μ broad. They show nucleus and blepharoplast, and may divide longitudinally before flagella are acquired (Figs. 2–4). The flagellum of each parasite arises from a region near to the blepharoplast but not directly from it (Figs. 5–9).

(5) The flagellate stage of the organism (Pl. V, Figs. 19–36) is that best known. H. jaculum is from 13μ to 33μ long and from 1μ to 4μ broad, the size varying according to the recency or otherwise of longitudinal division. Myonemes (Figs. 20, 21, 38) are present on the body. The flagellum is at least as long again as the body. The nucleus contains a number of grains of chromatin (Figs. 28, 34), sometimes in the form of eight large grains (Figs. 34, 36), sometimes as very fine granules (Figs. 22, 31). The blepharoplast is in the anterior, pre-nuclear, region of the parasite, and is usually rod-like (Figs. 19, 20, 21). The single flagellum (Fig. 19) arises near it but not from it. A basal granule (Figs. 22, 33, 34) is present at or near the origin of the flagellum. Chromidia are present as scattered granules in the body (Figs. 28, 32, 33).

(6) The post-flagellate stage is the form assumed by the parasite for life outside the body of the host. Preceding encystment, the organism divides twice longitudinally, giving rise to four daughter forms (Pl. V, Figs. 51–54) each of which ultimately loses its flagellum, rounds itself off and forms a cyst (Figs. 57–68). These cysts are from 2·5μ to 4·5μ long and from l·4μ to 2·6μ, broad. They occur in the rectum of Nepa cinerea and are voided with the faeces, being ingested later by other bugs.

(7) Longitudinal division is the common method of multiplication of H. jaculum. The flagellum may divide precociously, but usually division is initiated by constriction of the blepharoplast (Pl. V, Fig. 37) almost simultaneously with division of the flagellum and followed by that of the nucleus (Figs. 39, 40). A split occurs (Figs. 40–42) and the active movements of the two flagella aid in the divergence of the daughter organisms (Figs. 43, 44), which ultimately separate.

(8) I have no evidence whatever for ascribing sex to any form of Herpetomonas, but consider the occurrence of long and short and of thin and stout forms to be explicable as the results of growth and division. Also, I have shown experimentally that richly granular protoplasm is the result of a physiological condition and is not necessarily fixed as an attribute of the female sex.

(9) One mode of infection has been proved experimentally in the laboratory and also observed at the breeding grounds of the Nepa. Cysts voided in infected faeces are swallowed by other Nepa in the adult and nymphal stages. The crops of such nymphs on dissection were found to contain cysts, whereas no flagellates were present in other parts of the gut, so that primary infection occurred here in the crops of the nymphs.

Cannibalism of Nepa cinerea whereby other Nepa are devoured is also responsible for the spread of H. jaculum.

I have no definite evidence of hereditary infection, although I have found flagellate and post-flagellate forms (Pl. V, Figs. 46–49) in the ovaries of the host. The parasites were not found in the eggs. The occurrence of parasites in the ovaries may be regarded as a stage in the evolution of hereditary infection.

(10) Various environmental effects have been studied, the most important observation being that fresh food appears to stimulate the parasites and to cause their rapid division.

(11) The generic name Herpetomonas should be retained, as originally constituted, for parasites having but one flagellum and no undulating membrane (see pp. 383 et seq.).

(12) I wish to record the occurrence of a new species of Herpetomonas, H. vespae, from the alimentary tract of the hornet, Vespa crabro.

Human neutrophils were infected with tachyzoites of Toxoplasma gondii in vitro. Infection rates after 1 h of incubation were 38·6–52·0% and 16·0–25·8% in the presence or absence of specific antibody respectively. Apparently, neutrophils killed tachyzoites 18 h after infection in the presence of antibody, whereas tachyzoites proliferated in 25·0–35·0% of infected neutrophils in the absence of antibody. Total tachyzoite counts 18 h after infection were approximately 4 times as high as those 1 h after infection in the absence of antibody and complement. Complement also enhanced phagocytosis and killing of tachyzoites by neutrophils, but the effects were less than that of antibody. Superoxide anion was produced intensively within 1 h after infection, depending on the presence of antibody or complement. Neutrophils seem to be responsible for dissemination of tachyzoites in the host during the initial phase of Toxoplasma infection until antibody is produced.

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.

In total, 61 Chilean isolates of Trypanosoma cruzi, were analysed using schizodeme and Southern analysis, using as probes the highly variable regions of minicircles from cloned parasites. Isolates were collected and amplified from domestic and wild triatomines, and from infected subjects in all the endemic areas of Chile. Three major parasite genotypes could be detected in the domestic transmission cycle, whilst 1 major T. cruzi genotype is circulating in the wild transmission cycle. Schizodeme analysis suggested that T. cruzi isolates are mixed populations, whereas the Southern analyses detected only 3 mixed isolates using 4 selected minicircle segments as probes.

The present study was undertaken in village Karondang in South-Sulawesi, Indonesia, to investigate the influences of genetic, household and environmental factors on Brugia malayi infection. Infection status was determined by measuring both microfilariae in night blood and anti-filarial IgG4, as a marker for detection of active filarial infection. A total of 171 residents participated in the study; familial relationships between subjects were registered to construct pedigrees and distances between households were measured. The data were analysed using a test statistic for familial aggregation. For distribution of microfilariae over the study population a genetic influence on infection susceptibility was favoured over the household and environmental effects. For anti-filarial IgG4, all 3 clustering models gave significant results, suggesting that genetic, household and/or environmental factors influence specific IgG4 antibodies.

Secretions of plant-parasitic nematodes which are released into plant tissue may play critical roles in plant-nematode interactions. The identification and characterization of these molecules are of fundamental importance and may help to facilitate the development of novel strategies to interfere with nematode infection of plants and thereby decrease nematode-induced damage to crops. An antibody-based approach was used to isolate molecules present on the nematode surface and in nematode secretions. Monoclonal antibodies (MAbs) were produced to secretions and to whole Heterodera avenue 2nd-stage juveniles; several of these MAbs recognized molecules present in nematode secretions produced in vitro. Three of these molecules have been partly characterized in H. avenae, Globodera rostochiensis, G. pallida and Meloidogyne incognita. A MAb reacting with the surfaces of these nematodes recognized antigens of different molecular weight in each of the species tested. This difference in antigenicity might be related to specific functions in these nematodes. Preliminary results show that this antibody also localized the antigen in root cells surrounding the feeding site induced by M. incognita in Arabidopsis thaliana.

Infection with Giardia lamblia often causes only minor mucosal changes to the small intestine yet frank fat malabsorption may still occur. Some evidence suggests abtext-abstract pancreatic exocrine function in subjects with giardiasis although the mechanism and significance of this is unclear. Studies were conducted in vitro to determine the effect of G. lamblia trophozoites or culture filtrates from the organism on lipolysis of triglyceride by porcine pancreatic lipase. Live trophozoites significantly inhibited lipolysis. The degree of inhibition increased with longer duration of lipase exposure to trophozoites. Total amounts of enzyme inhibited were proportional to enzyme concentration, while the percentage inhibition was greatest at lowest concentration. At a lipase concentration of 1·7 i.u./ml, enzyme activity was reduced by 89·7% compared to controls after incubation for 4 h with trophozoites. The effect was abolished using killed, intact trophozoites. Culture filtrates of G. lamblia did not inhibit lipolysis. Specificity of the effect was suggested by the failure of another flagellate protozoan, Trichomonas vaginalis, to inhibit lipase. In this assay system the inhibition of lipolysis was not dependent on the bile salt concentration present. The impact of this effect in vivo remains to be determined but it may contribute to fat malabsorption in giardiasis.

Toxocara canis infective larvae are known to produce abundant glycosylated molecules which may be found associated with the surface or secreted into their environment. Using a range of fluorescein-conjugated and gold-conjugated lectins, the localization of particular carbohydrates was defined on the surface of live parasites, and internally at the ultrastructural level. Surface exposure of N-acetyl galactosamine and N-acetyl glucosamine was deduced by binding of FITC-conjugated Helix pomatia (HPA) and wheat-germ agglutinins (WGA). These sugars appear to be associated with a densely staining surface coat as conventional immuno-electron microscopy procedures dissipate this coat and reveal no surface binding site for these lectins. However, by using cryo-immuno-electron microscopical (C-IEM) techniques, the surface coat is retained and can be shown to bind WGA. The fluorescent lectins also revealed strong WGA binding to the secretory and amphidial pores, while the buccal opening and the cuticular alae bound HPA. Corresponding results were obtained at the ultrastructural level. Thus, HPA bound to the electron-dense area of the cuticle, areas of local cuticular thickening such as the alae and buccal labia, as well as to the oesophageal lumen. WGA also bound to the thickened cuticle of the alae and the buccal opening, but showed no reaction to either the electron-dense layer of the cuticle or the oesophageal lumen. Unlike HPA, WGA did bind specifically to the secretory column contents and the electron-dense regions of the lips associated with the chemosensory amphids. The compartmentalization of the sugars N-acetyl galactosamine and N-acetyl glucosamine, their sources and routes of surface expression and the possible association with the TES glycoprotein antigens are discussed.

This study investigated the molecular and biological variation among different Giardia duodenalis assemblages. In vitro growth and susceptibility to albendazole, fenbendazole, flubendazole, metronidazole, tinidazole and furazolidone was studied for laboratory (AI: WB, AII: G1 and B: GS/M-83-H7) and 6 field isolates of assemblage subtype AI, AII, B and EIII. Additionally, isolates of the 3 assemblages were evaluated in the gerbil upon 3-day oral treatment with albendazole (6 mg/kg), flubendazole (5 mg/kg) and metronidazole (20 mg/kg). Assemblage AI grew significantly faster than all other assemblage subtypes, which showed comparable generation times. The assemblage A laboratory strains displayed altered in vitro drug susceptibilities compared to their matching AI or AII field isolate. No variation in drug susceptibility was observed between field isolates of assemblages A and E. However, assemblage A laboratory strains were more susceptible to the benzimidazoles and less susceptible to the nitro-imidazoles and furazolidone than the assemblage B laboratory strain. In the gerbil, no markedly different drug susceptibilities were observed. In conclusion, the Giardia assemblage subtype can be associated with differences in growth characteristics rather than in drug susceptibility.

The number of schistosomula in lungs was determined by compressed organ autoradiography at intervals up to 14 days after exposure of mice to 75Se-labelled cercariae by tail immersion. Probit analysis of compressed lung autoradiogram focus counts, expressed as percentages of initial infection level, yielded estimates of the average time of arrival, peak accumulation in the lungs and average time of departure of schistosomula: 4·5±0·87, 6·3 ± 0·45 and 11±0·58 days, respectively. At peak accumulation 92±3·5 % of the initial number of schistosomula were found in the lungs. It thus appears that little or no significant attrition of schistosomula occurred in the skin and, instead, that most of the 50–70% of penetrant cercariae that fail to reach adulthood are lost somewhere between the pulmonary and hepatic phases of development. Loss of 75Se label from schistosomula during the first 14 days was exponential, with an average half-life of 4·5±0·81 days. However, the high sensitivity of autoradiography tended to compensate for this rather rapid rate of label loss. It was pointed out that autoradiographic detection of schistosomula as discrete loci of radioactivity can also be expected to overcome the problem posed by the accumulation in such tissues as liver and kidney of 75Se label that has become separated from larvae.