Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic tool for M. sydneyi.

The liver fluke, Fasciola hepatica relies on a well-developed muscular system, not only for attachment, but for many aspects of its biology. Despite this, little is known about the system beyond the gross organization of the main somatic muscle layers. In the present study, a range of techniques have been applied to F. hepatica in order to understand more about various aspects of muscle organization, biochemistry (in terms of muscle proteins) and identity of isolated muscle fibres. Scanning electron microscopy has provided a direct visualization in situ of the somatic muscle layers and the organization of the muscle fibres within the ventral sucker. The muscle bundles contributing to the main somatic muscle layers are made up of up to 10 individual muscle fibres. Phalloidin staining for actin, in conjunction with confocal microscopy, confirmed the presence of 2 main somatic muscle layers (outer circular, inner longitudinal), beneath which lies a third layer of oblique muscle fibres. The use of propidium iodide in combination with phalloidin staining for actin demonstrated that the cell bodies associated with the 2 main somatic muscle layers are situated beneath the longitudinal muscle layer and are connected to their respective muscle fibres by short cytoplasmic processes. Myosin immunoreactivity was demonstrated in the somatic muscle layers and in the muscle layers surrounding various organ systems within the fluke. Double labelling for actin and myosin confirmed the co-localization of the 2 muscle proteins in the muscle fibres of the ventral sucker. Muscle fibres from the somatic muscle layers and the ventral sucker have been isolated and images obtained with phase-contrast microscopy and scanning electron microscopy. The muscle fibres contain actin and myosin, but lack a nucleus, the connection with the cell body having been broken during the isolation procedure.

The questing height (i.e. ambush height) of ticks on a plant plays an important role in host selection. To test the hypothesis that the questing height of ticks in a locality had adapted to the body size of the host in that locality, we examined the questing height of nymphs of the ticks, Haemaphysalis longicornis and H. mageshimaensis, at 7 locations in Japan. Sika deer, Cervus nippon, is the primary host of these ticks and there is considerable geographical variation in the body size of sika deer. Multiple regression analysis revealed that the questing height in the field was influenced by the height of the plants and by the body size of deer at a location. However, the questing height of ticks at some locations may have been constrained by the height of the plants and might not be the same as their intrinsic questing height. When ticks were placed in vertical glass tubes in the laboratory, the questing height of ticks from a locality was correlated with the mean body size of deer at that locality. Therefore, the prominent cue determining the questing height of H. longicornis and H. mageshimaensis seems to be the body size of the host deer.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

The Trypanosoma brucei metacyclic population in the salivary glands of the tsetse fly displays a characteristic set of variable antigen types (VATs) which represents only a restricted part of the parasite's total VAT repertoire. After introduction into the mammalian host by fly bite, the metacyclics transform into bloodstream forms which retain expression of the metacyclic VATs. Specific antibodies, both polyvalent and monoclonal, have been used to neutralize separately 4 individual VATs from metacyclic populations. Control experiments and visual observation confirmed lysis of each VAT. On injection of the surviving trypanosomes, after washing, into mice each neutralized VAT was nevertheless expressed within a few days. Simultaneous neutralization of 2 metacyclic VATs which usually switch to one another in bloodstream infections did not prevent expression of either on subsequent injection into mice. Expression of neutralized VATs was not influenced by the antigenic composition of the population originally ingested by the tsetse fly. Metacyclic forms and their immediate successors thus appear to switch rapidly to expression of other metacyclic VATs in bloodstream populations.

The effects of duration of infection and of temperature and nourishment in cultures on development of free-living generations of Strongyloides stercoralis were studied quantitatively. Rhabditiform larvae, 228–353 μm long, were collected from infected dogs with or without prednisolone treatment using the Baermann apparatus. Cultures were carried out by the filter paper test-tube method under the following conditions: incubation temperature 15–40°C and faecal dilution 1:0–1:16. Rhabditiform larvae developed predominantly to free-living females at incubation temperatures of 15–30°C and low faecal dilutions, but filariform larvae appeared mainly under extreme conditions such as high temperature. Recovery rates of filariform larvae were inversely related to those of females. It was remarkable that high temperature, but not low faecal dilution, affected development of filariform larvae. Although the appearance of free-living males was constant in various environmental conditions, the present study indicated an increase in free-living males with the duration of infection. Thus, it seems that free-living males are already fixed as male in the egg stage, and potential female rhabditiform larvae differentiate into free-living females or filariform larvae depending on environmental factors. There is no marked difference in the development of rhabditiform larvae into filariform larvae in either the immunosuppressed dog or the intact dog.

Rohdella siamensis, a new genus and species of aspidogastrean, is described from freshwater fishes in Thailand. A new subfamily, the Rohdellinae, is erected to accommodate this worm on the grounds that it differs from all known aspidogastreans in that the male and female ducts unite to form an hermaphroditic duct, with the result that the intromittent organ is a sinus-organ and not a cirrus. A new classification of the subclass Aspidogastrea is presented. Two orders are recognized: the Aspidogastrida, containing a single family, the Aspidogastridae, which in turn contains 3 subfamilies, the Aspidogastrinae, the Cotylaspidinae and the Rohdellinae subfam.nov.; and the Stychocotylida ord.nov., containing 3 families, the Stichocotylidae, the Multicalycidae fam.nov. and the Rugogastridae. The systematic position of the Stichocotylida, and especially the Rugogastridae, is commented upon.

Adult specimens of Trochopus pini attach themselves by folding the disk-shaped haptor around the edge of the host's primary gill lamella. They are able to move from one place to another with the aid of disk-shaped pads on the head region.Ducts from the posterior median gland cells, which are extensive in the head region of the adult, open on the periphery of each disk and exude an adhesive secretion.

The oncomiracidium of T. pini is described and evidence is offered in support of direct invasion of the gills by these free-swimming larvae. Special attention is paid to the development of the head region of the parasite. Newly attached larvae lose their eye lenses and possibly their anterior median gland cells; their lateral head glands are well developed and most probably serve for attachment of the head region, since the posterior median gland cells are poorly developed and appear to have no ducts. Later the posterior median gland cells and their ducts increase in numbers and, in association with the pads on the head region, take over from the lateral glands the job of attaching the head region.

The paths of the tendons associated with the accessory sclerites on the haptor of T. pini indicate that this gill parasite has evolved relatively recently from the ancestors of benedeniid skin parasites.

I am most grateful to Commander J. Pareira, the Director of the Aquario Vasco da Gama in Lisbon where most of this work was done, and to the Staff of the Aquario and especially to Mr A. Ferreira. I am indebted to the Calouste Gulbenkian Foundation for a generous grant which made this visit possible. I am grateful also to the Directors and Staff of the Laboratoire Arago, Banyuls, France, the Station Biologique of the University of Bordeaux at Arcachon in France, and the Laboratory of the Marine Biological Association of the United Kingdom at Plymouth, England. I would like to thank the Royal Society for a grant from the Browne Fund which enabled me to work at Banyuls in 1962 and for a Pergamon Travel Award which made it possible to visit Arcachon in 1967.

The pathogenic flagellate Blastocrithidia triatomae disrupts the digestion of Triatoma infestans; the midgut ultrastructure of bugs infected with the flagellate and of uninfected bugs is compared. Third or fourth instar larvae were dissected either unfed or 1 week after feeding.In all uninfected bugs extracellular membrane layers (e.m.l.) covered the apical microvillar border of the epithelial cells. Some midgut regions of bugs infected with B. triatomae appeared normal but often adjacent cells showed pathological effects. In affected cells the e.m.l. and the microvilli and finally the cells themselves were reduced or destroyed. Correlated with these observations of pathogenicity the method of attachment of parasites changed. When the e.m.l. were present only rarely were flagella found, but on extracellular membrane-free cells B. triatomae attached by flagellar enlargement to the microvillar border or, if this was reduced, to the apical host cell membrane. No hemidesmosome-like plaques were found at the attachment site. Although some flagella were inserted into the apical region of the cells no intracellular flagellates were observed.

The mode of action of suramin upon Brugia pahangi has been investigated in vivo and in vitro. The drug was without effect on the glycolytic activity of worms in vitro at 2 × 10−4 M. The lack of effect was correlated with the failure of [14C]suramin to penetrate the worms in vitro. Suramin bound to the surface of worms in vitro presumably by virtue of its polyanionic nature. B. pahangi adults ingested [14C]suramin in vivo but no reduction in the rate of lactate production, of glucose utilization or in the rates of uptake of [14C]glucose, [14C]leucine or [14C]adenosine was observed in worms recovered from jirds between weeks 1 and 5 following 4 daily doses of suramin at 50 mg/kg given intraperitoneally. Worm death occurred between weeks 5 and 7 but this delayed drug effect was not the result of a progressive accumulation of suramin in the worms. Ultrastructural changes were observed in the intestinal epithelium of worms from suramin-treated jirds and parallel observations on worms exposed to Trypan blue in vivo suggest that both polyanionic compounds are restricted to the intestinal lumen of the worms. The evidence presented is consistent with the concept that, in B. pahangi, suramin acts at the surface of the intestinal epithelium and not by primarily inhibiting glucose catabolism or inhibiting phagosome and lysosome fusion as previously demonstrated for bloodstream trypanosomes and mammalian macrophages, respectively

Density-dependence in worm establishment, numbers, biomass and larval production were examined in primary infections of 0, 10, 40, 80 and 160 larvae of the lung nematode, Rhabdias bufonis in the common toad, Bufo bufo. The infection procedure established 4 non-overlapping levels of infection which persisted until 6 weeks post-infection (p.i.), after which there was an overall decline up to 12 weeks p.i. Worm numbers had no direct effect on adult worm survival but temporal changes in worm weight were density-dependent. Adult worm establishment in the lungs declined significantly as the numbers of worms in the lungs increased. At the lowest exposure dose, 86% of the larvae administered reached maturity in the lungs while at the highest, only 37% did so. Also, the numbers of immature larvae outside the lungs increased as adult worm numbers increased. Both features provide evidence for a threshold limit to the numbers of worms maturing in the lungs. Worm numbers also affected larval output per host and per capita fecundity. A significant positive relationship between per capita fecundity and per capita worm weight suggested that density-dependence acted primarily to constrain the growth of individual worms. Finally, the constraints imposed on worm growth and fecundity were apparently relaxed when worm density decreased, providing evidence for density-dependent flexibility in per capita fecundity. Density-dependence in worm establishment and per capita fecundity are mechanisms which may potentially regulate this host–parasite interaction in the field. Both mechanisms may be functionally related to physical space limitations in the lungs, within which worms must compete for finite nutrients.

‘Each organism must be examined as a biochemical entity before any reasonable understanding of helminth metabolism can be attained’ (Saz, 1969).

The purpose of this work is to review the progress towards malaria vaccination that has been made over the last four or so years, and the prospects and difficulties as they now appear. Although some of the older literature will be referred to as necessary background, it is not treated here in any detail. The reader who wishes for a fuller historical perspective should see, for instance, Brown (1969), Cohen & Mitchell (1978), Desowitz & Miller (1980), Mitchell (1984), Miller, David & Hadley (1984), Heidrich (1986) and, specifically for a consideration of sporozoite vaccination, Nussenzweig & Nussenzweig (1984, 1986). Naturally, any summary of vaccination will lean on the immunology of the disease, but malaria immunology is not reviewed here in its own right. The reader requiring the most recent attempt to cover this field should see the work edited by Perlmann & Wigzell (1988); some individual chapters of that volume will be referred to below.

Experiments were carried out to show the different hygroscopic properties of certain foods supplied to flea larvae. These show that larvae of Ceratophyllus wickhami require about 15 to 28 per cent, of water in their food. Dried blood, albumen (blood) and material from the bottom of a squirrel's nest were used.

Newly hatched flea larvae were fed on different substances at varying humidities and a constant temperature. A relative humidity of 80 per cent. gave the best results.

Dried blood is a convenient food for breeding flea larvae in the laboratory, and may be used with or without a mixing medium, such as sand. Albumen is a more simple food on which a few larvae will survive, but at lower humdities than dried blood.

Flea larvae will eat a great variety of other foods, including squirrel faeces, ground-up cockroach and dead adult fleas, but these do not provide a complete diet.

It does not seem necessary for the larvae to have dried blood from the host of their parents, rabbit or human blood being quite as satisfactory.

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42°C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis.

Various treatments are found to be moderately effective in managing Demodex-related diseases except tea tree oil (TTO) and terpinen-4-ol (T4O), which showed superior miticidal and anti-inflammatory effects in numerous clinical studies. Their possible effects include lowering mite counts, relieving Demodex-related symptoms, and modulating the immune system. This review summarizes the current clinical topical and oral treatments in human demodicosis, their possible mechanisms of action, side-effects and resistance in treating this condition. TTO (especially T4O) is found to be the most effective followed by metronidazole, ivermectin and permethrin in managing the disease. This is because TTO has anti-parasitic, anti-bacterial, anti-fungal, anti-inflammatory and wound-healing effects. Furthermore, nanoTTO can even release its contents into fungus and Pseudomonas biofilms. Combinations of different treatments are occasionally needed for refractory cases, especially for individuals with underlying genetic predisposal or are immuno-compromised. Although the current treatments show efficacy in controlling the Demodex mite population and the related symptoms, further research needs to be focused on the efficacy and drug delivery technology in order to develop alternative treatments with better side-effects profiles, less toxicity, lower risk of resistance and are more cost-effective.

Allelic variation in 6 microsatellite markers was compared between frozen Schistosoma mansoni eggs and laboratory-passaged worms originating from the same 5 fecal samples obtained from Brazilian residents. Based on allelic richness values, the number of alleles detected per locus did not differ between egg and worm DNA templates. However, our ability to score loci differed between these DNA templates, with worms providing more scored loci per individual than eggs. Differences also existed between the worms and eggs in the identity of the specific alleles that were detected. Additionally, we observed a reduction in homozygous genotypes among laboratory-passaged worms relative to the eggs. Allelic diversity curves were calculated by genotyping all worms from a representative host sample to determine the relationship between the number of alleles detected at a locus and the number of worms genotyped. Curves for the 5 residents' worm infrapopulations for each of the loci were very similar. The equation y=19·55×ln(x)+9·992 explained the association between sampling effort (x) and number of alleles detected (y) with an R2 of 0·775. In conclusion, egg DNA templates and allelic diversity curves can benefit efforts to discern the sociological, ecological and evolutionary forces impacting the genetic diversity and disease epidemiology of human schistosomes.

Due to the low informative value of available morphological characters, cytogenetic and molecular methods, based on rDNA sequencing, were used to characterize adult and larval stages of Phyllodistomum spp. Species studied have 18 chromosomes with comparable absolute and relative lengths. Conventional Giemsa staining and karyometric analysis revealed clear differences in chromosome morphology of larval Phyllodistomum spp. infecting two bivalve host species, Sphaerium corneum and Pisidium amnicum. However, karyotypes of adult P. folium from three-spined sticklebacks and larval stages from S. corneum appear almost identical both with respect to the relative lengths and centromeric indices of the corresponding chromosome pairs. The entire internal transcribed spacer (ITS) region (ITS-1, 5.8S and ITS-2) and the D1-D3 region of 28S gene were sequenced and compared. Again, sufficient differences were observed between larval Phyllodistomum spp., while adult P. folium and larvae from S. corneum showed a high level of similarity. So, both cytogenetic and molecular data support the suggestion that they represent developmental stages of the same species. The results were compared with published data obtained by cytogenetic and molecular studies on the other Phyllodistomum species. Differences revealed in karyotype and rDNA sequences leads to the conclusion that the cercariaeum of P. folium sensu Sinitsin, 1905 could not be regarded as the larva of adult P. folium from three-spined stickleback.

This study examines polymorphisms in 2 genes (pfmdr1 and cg2), which have been associated with resistance to chloroquine in Plasmodium falciparum, to determine their value as predictors of resistance status. Among field samples from children in Zaria, northern Nigeria, the Tyr-86 polymorphism in pfmdr1 and Ala-281 and the Dd2κ repeat of cg2, were significantly associated. In 8 samples classified resistant by the micro-in vitro test, or, where this failed, by in vivo trial, 7 showed the cg2 Dd2 type κ repeat, and 6 of these had both the Ala-281 allele and the pfmdr1 Tyr-86 allele. In 26 chloroquine-sensitive samples, none had this combination of 3 polymorphisms (P = 0·00002). This indicates 75% sensitivity and 100% specificity in detection of resistance and shows a positive predictive value for resistant infections of 100%. The negative predictive value, because of sensitivity less than 100%, would depend on the prevalence of resistance. Where prevalence of resistance is approx. 21% as in Zaria, the negative predictive value would be 94%, while in Gabon, with a prevalence of ca 73% it would be 60%. The use of (cg2: Ala-281, Dd2κ. pfmdr1: Tyr-86) genotype detection as a predictive epidemiological tool to examine the distribution of chloroquine-resistance in parts of Africa is therefore possible. The sensitivity of detection of resistant strains still requires improvement.

Polistes dominulus are social wasps which are the host of the strepsipteran endoparasite Xenos vesparum. In the hibernating phase, unparasitized and parasitized wasps leave natal nests and aggregate together in sheltered quarters. In aggregations, wasps are socially active, and some individuals perform helping behaviour. Here we investigated if castrated parasitized wasps perform worker tasks in mixed aggregations. Moreover, by gas chromatography and mass spectrometry, we examined the cuticular hydrocarbons of unparasitized and parasitized wasps to evaluate if the infection alters the composition of cuticular waxes that are recognition cues in social insects. In clusters, infected females do not perform helping behaviour and they are less active than unparasitized wasps. Cuticular hydrocarbons are slightly differentiated between unparasitized and parasitized wasps but, generally, unparasitized wasps are more similar to wasps infected by Xenos females compared to wasps infected by Xenos males. Wasps infected by Xenos males do not usually survive the winter. This chemical similarity is probably a consequence of the similar physiological condition of unparasitized and female-affected Polistes wasps. At this stage, it is difficult to affirm whether these modifications are a true parasite manipulation or a consequence of infection.