Serology is an important tool for the diagnosis of alveolar echinococcosis (AE) in humans. In order to improve serodiagnostic performance, we have developed an in vitro-produced Echinococcus mulilocularis metacestode vesicle fluid (EmVF) antigen for application in an immunoblot assay. Immunoblot analysis of EmVF revealed an abundant immunoreactive band triplet of 20–22 kDa, achieving a sensitivity of 100% based on the testing of sera from 62 pre-operative and pre-treatment cases of active and inactive AE. Thus, the EmVF-immunoblotting allowed the specific detection of cases seronegative by the Em2- and/or EmII/3–10-ELISA, usually attributable to abortive, inactive cases of AE. The specificity of the EmVF-immunoblotting did not allow discrimination between AE and cystic echinococcosis (CE) but was 100% with respect to non-Echinococcus parasitic infections or cancer malignancies. Based on the findings of this study, it is recommended that the current ELISA test combination (Em2- and II/3–10-ELISA) be complemented with EmVF-immunoblotting, allowing an improved diagnosis of both clinical and subclinical forms of AE, including those associated with E. multilocularis-specific antibody reactivities not detectable by ELISA.

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1×10−5m) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1×10−6m), a sodium ionophore and ammonium chloride (NH4Cl) (5×10−2m), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1×10−3m) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

The reactions of the house-fly larva to four environmental factors, namely temperature, air humidity, smell and light have been studied in the Laboratory. It was hoped that the results would shed some light on the general behaviour of the larva in its normal habitat. A comprehension of this behaviour will assist in understanding the ecology of the species, and hence facilitate its control. In order to analyse the behaviour of the larva, its reactions (other than to light) were first studied in relation to one factor at a time. An attempt was then made to test experimentally its reactions to various combinations of factors.

Perforated plastic cylinders (hair curlers) were implanted subcutaneously in rabbits for the collection of tissue fluid. Using this technique, a study was made of the chemical constituents of tissue fluid and serum in rabbits infected with Trypanosoma (Trypanozoon) brucei, as compared with uninfected, control animals. Changes were caused by the infection in the concentrations of proteins, electrolytes, urea, creatinine, cholesterol and pyruvate, and in the activities of the enzymes aspartate transaminase, creatine phosphokinase and alkaline phosphatase. Antitrypanosomal antibody, detectable by the indirect fluorescent antibody technique, was shown to penetrate the tissue fluid and to reach a titre of about one-fifth of that in the serum. Renal failure, probably resulting from allergic reactions, was shown to be a contributory cause of death in rabbits infected with T. (T.) brucei.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ixodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 °C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.

Tsetse flies (Glossina morsitans) were fed on the blood of mice containing any one of 5 variable antigen types (VATs) of Trypanosoma brucei AnTAR 1 serodeme. The VATs of the metacyclic trypanosomes subsequently detected in the flies' saliva probes were investigated using monospecific antisera to AnTAR 1 VATs in indirect immunofluorescence and trypanolysis reactions; these sera included 3 raised against AnTats 1.6, 1.30 and 1.45, previously identified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6·0–8·3%; AnTat 1.30, 13·7–18·2%; AnTat 1.45, 2·0–8·0%), regardless of the age of the fly or the ingested VAT. As these 3 VATs accounted for no more than 30% of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes.

1. The enzymic activity was studied in the following Dermatophyte fungi: Sabouraudites radiolatus; S. lanosus; S. audouini; Trichophyton tonsurans; Grubyella schoenleinii. Both the normal and pleomorphic forms of S. radiolatus were studied comparatively.

2. An active proteolytic enzyme is present in all the species. This enzyme is active in an alkaline medium and can hydrolyse intact proteins (casein) with the production of free amino-acids (tryptophane), and is very similar to trypsin in its behaviour.

3. Pepsin is not present in any of these fungi.

4. The amount of the proteolytic enzyme present varies in the different species, and it is particularly abundant in S. radiolatus.

5. A keratolytic enzyme was not found in any of these fungi.

6. A lipolytic enzyme, lipase, which readily splits tributyrin into fatty acids, is present in all the species. It is about equally strong in all of them.

7. Urease is present in all, with the exception of T. tonsurans.

8. None of them contains invertase, inulase, lactase or zymase.

9. Maltase and diastase are present in all. They are strongest in S. lanosus and T. tonsurans and are weakest in S. radiolatus.

10. The species with most proteolytic activity have least carbohydrases, and conversely.

11. Amygdalase is present in all the species.

12. The normal form of S. radiolatus has greater proteolytic activity than the pleomorphic form. The pleomorphic form has strong urease and amygdalase, both of which are very weak in the normal form. Otherwise the enzymic activity of the two forms is similar.

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

Leishmaniases is a tropical disease caused by protozoa of the genus Leishmania for which the current treatment is expensive, besides increasing reports of parasite resistance. This study investigated the anti-Leishmania amazonensis activity of the essential oil from Aloysia gratissima (AgEO) and guaiol, the major sesquiterpene constituent in the oil. Our results showed that AgEO killed promastigotes and intracellular amastigotes at an IC50 of 25 and 0·16 µg mL−1, respectively, while guaiol killed amastigotes at an IC50 of 0·01 µg mL−1. Both AgEO and guaiol were safe for macrophages up to 100 µg mL−1, as evaluated by the dehydrogenase activity, membrane integrity and phagocytic capacity. AgEO and guaiol did not induce nitrite oxide (NO) in resting macrophages and inhibited the production of NO in lipopolysaccharide-stimulated macrophages. The ultrastructural analysis suggested that AgEO and guaiol act directly on parasites, affecting promastigotes kinetoplast, mitochondrial matrix and plasma membrane. Together, these results pointed out that AgEO and guaiol could be promising candidates to develop anti-Leishmania drugs.

Virulent strains of Entamoeba histolytica have been reported to produce a mixture of phosphoglycoconjugates that share some structural features with the lipophosphoglycans (LPGs) of Leishmania. Purification of these glycoconjugates is essential to their precise structural characterization. In this study we have extracted ‘LPG-like’ molecules from various virulent E. histolytica strains and purified on the basis of charge differences, 2 apparently related glycoconjugates a ‘LPG’ and a ‘lipophosphopeptidoglycan (LPPG)’. In marked contrast to the abundance of these ‘LPG’ and ‘LPPG’ molecules in the virulent strains, avirulent E. histolytica and E. dispar strains produce either very low, or no detectable levels of LPG, and either low levels or modified forms of ‘LPPG’. Monospecific polyclonal antibodies prepared against that ‘LPG’ of the virulent strain HM-1: IMSS cl6 identified epitopes shared between both the ‘LPG’ and the ‘LPPG’ of this and other virulent strains, using Western blot analysis. Flow cytometric analysis of a range of strains using these antibodies identified a surface distribution of these molecules and confirmed a correlation between surface exposure of epitopes bound by these antibodies and parasite virulence.

The public health threat posed by zoonotic Plasmodium knowlesi appears to be growing: it is increasingly reported across South East Asia, and is the leading cause of malaria in Malaysian Borneo. Plasmodium knowlesi threatens progress towards malaria elimination as aspects of its transmission, such as spillover from wildlife reservoirs and reliance on outdoor-biting vectors, may limit the effectiveness of conventional methods of malaria control. The development of new quantitative approaches that address the ecological complexity of P. knowlesi, particularly through a focus on its primary reservoir hosts, will be required to control it. Here, we review what is known about P. knowlesi transmission, identify key knowledge gaps in the context of current approaches to transmission modelling, and discuss the integration of these approaches with clinical parasitology and geostatistical analysis. We highlight the need to incorporate the influences of fine-scale spatial variation, rapid changes to the landscape, and reservoir population and transmission dynamics. The proposed integrated approach would address the unique challenges posed by malaria as a zoonosis, aid the identification of transmission hotspots, provide insight into the mechanistic links between incidence and land use change and support the design of appropriate interventions.

Deer are important blood hosts for feeding Ixodes ricinus ticks but they do not support transmission of many tick-borne pathogens, so acting as dead-end transmission hosts. Mathematical models show their role as tick amplifiers, but also suggest that they dilute pathogen transmission, thus reducing infection prevalence. Empirical evidence for this is conflicting: experimental plots with deer removal (i.e. deer exclosures) show that the effect depends on the size of the exclosure. Here we present simulations of dynamic models that take into account different tick stages, and several host species (e.g. rodents) that may move to and from deer exclosures; models were calibrated with respect to Ixodes ricinus ticks and tick-borne encephalitis (TBE) in Trentino (northern Italy). Results show that in small exclosures, the density of rodent-feeding ticks may be higher inside than outside, whereas in large exclosures, a reduction of such tick density may be reached. Similarly, TBE prevalence in rodents decreases in large exclosures and may be slightly higher in small exclosures than outside them. The density of infected questing nymphs inside small exclosures can be much higher, in our numerical example almost twice as large as that outside, leading to potential TBE infection risk hotspots.

To investigate the phylogeny of benign Theileria parasites, we determined the complete major piroplasm surface protein (MPSP) gene sequences for 6 benign theilerial organisms, including the first from tick. Sequences were analysed alongside published sequences for 39 benign Theileria parasites, using Bayesian inference and maximum parsimony. All MPSP sequences were 852 nucleotides, except for Gansu, Wuchangbuf, VB01, and VB01; Gansu contained 873 nucleotides, and the other 3 had 855. Deduced amino acid sequences contained 284 residues, except for Gansu (291) and Wuchangbuf, VB01, and VB01 (285 each). Pairwise comparisons showed identities among 45 theilerial MPSP sequences ranging from 70·9 to 99·8% for nucleotide and 71·0 to 100% for amino acid sequences. Our results clearly indicate that all global parasites, excluding Brisbane, were classified into 1 of 8 types; 6 types of Theileria exist in Korea. Each type, excluding Type 6, has several type-specific amino acid sequences. The phylogenetic tree derived from the nucleotide sequences showed 2 sister-group relationships, Type 2+Type 7 and Type 3+Brisbane, with a new branching pattern: (Type 6 (Type 8 ((Type 2, Type 7), (Type 1, (Type 4, (Type 5, (Type 3, Brisbane))))))). Our sequence data showed no geographical influence on worldwide Theileria parasite distribution.

Phenotypic alterations induced by parasites in their intermediate hosts often result in enhanced trophic transmission to appropriate final hosts. However, such alterations may also increase the vulnerability of intermediate hosts to predation by non-host species. We studied the influence of both infection with 3 different acanthocephalan parasites (Pomphorhynchus laevis, P. tereticollis, and Polymorphus minutus) and the availability of refuges on the susceptibility of the amphipod Gammarus pulex to predation by 2 non-host predators in microcosms. Only infection with P. laevis increased the vulnerability of amphipods to predation by crayfish, Orconectes limosus. In contrast, in the absence of refuges, the selectivity of water scorpions, Nepa cinerea, for infected prey was significant and did not differ according to parasite species. When a refuge was available for infected prey, however, water scorpion selectivity for infected prey differed between parasite species. Both P. tereticollis- and P. laevis-infected gammarids were more vulnerable than uninfected ones, whereas the reverse was true of P. minutus-infected gammarids. These results suggest that the true consequences of phenotypic changes associated with parasitic infection in terms of increased trophic transmission of parasites deserve further assessment.

Stability of trichostrogylid populations indicates that some form of density-dependent regulation occurs which could act through fecundity. We present evidence for intraspecific density-dependent effects in 1 of 2, dominant, abomasal nematodes species (Ostertagia gruehneri) of Svalbard reindeer (Rangifer tarandus platyrhynchus). We found evidence in O. gruehneri, for density-dependent regulation of female worm length in April, July and October 1999. However, it is only in July that female worm length explains the variation in the number of eggs in utero which is also related to egg production per female worm only in this month and not at other times of the year. The seasonal pattern in faecal egg output in this species focuses egg production in the summer months when conditions are favourable to transmission. In contrast, we found no evidence in the other common species (Marshallagia marshalli) for density-dependent regulation of female worm length during or the number of eggs in utero. Faecal egg output in M. marshalli was positively related to worm burden but not to the mean number of eggs in utero. Neither inter-specific interactions nor host body condition appeared to influence worm fecundity. The contrasting patterns of density-dependent regulation of fecundity provides further evidence for divergent life-histories in this nematode community.

It has been shown that mating involving mutual exchange of spermatophores takes place in the skin-parasitic monogenean Entobdella soleae from Solea solea. The spermatophores are attached to the ventral surface of the body in the region of the vaginal opening and they are sucked into the vagina after mating by muscular contractions of the vaginal region.

Mating takes place between young individuals with no vitellaria and between fully mature egg-laying adults but there is no evidence that self-fertilization takes place.

The apparatus which manufactures the spermatophores is described and the evidence indicates that spermatozoa are injected into the jelly-like matrix of the spermatophore as the matrix is being extruded into the surrounding sea-water.

I would like to thank the Director and Staff of the Laboratory of the Marine Biological Association at Plymouth for providing excellent facilities for this work and I am particularly grateful to Mr J. E. Green, who kindly fed and maintained infected soles at the Laboratory.

The adhesive organs of diplectanid, tetraonchid and dactylogyrid monogeneans develop in very similar ways indicating that all these gill parasites are closely related to each other. The oncomiracidia make initial contact with the general body skin or the lining of the buccal cavity to which they attach themselves by 14 or 16 ventrally orientated marginal hooklets. The growing parasites then find their way to the gills which call for a different kind of adhesive apparatus. In order to anchor itself between two of the host's secondary gill lamellae, two pairs of the parasite's marginal hooklets migrate to the dorsal surface of the haptor and two of the four developing hamuli rotate so that they also point dorsally.

In diplectanids the hamuli are assisted by accessory friction pads (squamodiscs) which develop on the surface of the body anterior to the hooks.

In dactylogyrids the ventral hamuli do not develop and are represented in the adult only by small spicules. In contrast the marginal hooklets grow during larval development and become important adult attachment organs, together with the dorsal hamuli.

Adults of Tetraonchus monenteron feed on the epidermis covering the gills.

I am most grateful to the Director and Staff of the Laboratory of the Marine Biological Association at Plymouth and particularly to Mr J. E. Green for help and hospitality. I would also like to express my thanks to the East Suffolk and Norfolk River Authority and particularly to the Fishery Superintendent, Mr E. Q. Bitton, and to Mr D. Leary, for supplying me with freshwater fishes.

1. The growth and development of normal and sterile (i.e. symbiont-free) Rhodnius prolixus are compared. The last three instars of sterile bugs weigh less than normal ones. Sterile insects completed the first three stages of their life-histories in less time than normal insects, and at the fourth stage, nutritional deficiencies had profound effects on their further development.

2. The sensitivity of the Rhodnius symbionts, Nocardia rhodnii, to five antibiotic substances is determined. Terramycin, aureomycin and chloramphenicol are effective inhibitors.

3. The symbiotic condition in Rhodnius and the related bug, Triatoma infestans, is discussed.

The effect of different relative humidities on the survival of individual larvae and adults of Ditylenchus dispasci and D. myceliophagus has been examined; experiments were carried out at 5 and 18°C with D. myceliophagusand at 18°C with D. dipsaci. Survival of both species was enhanced when they were dried at high humidities and, for D. myceliophagus, by the lower temperature. The survival of all stages of D. myceliophagus was poor. Generally it was expressed in minutes only; 4th-and 3rd-stage larvae, however, were marginally better at surviving drying than adults and much better than 2nd-stage larvae.

Although the 4th-stage larva of D. dipsaci was overwhelmingly superior to all other stages, the 3rd stage, and to a lesser extent, the adult of this species also showed remarkable powers of survival to desiccation. In general, survival of 4th-, 3rd- and 2nd-stage larvae could be expressed in weeks, days and minutes respectively; for adults, survival was expressed in hours at relative humidites under 50% but in days at higher relative humidites.

The results are discussed in relation to the survival value of the aggregations formed by these species; the validity of the term ‘resistantstage’ as applied to the 4th-stage larva of D. dipsaci is assessed.