We tested a theoretical model based on the physics of capillary flow and confirmed that anaemia accelerates blood intake in the yellow-fever mosquito, Aedes aegypti (L.). We also investigated the influence of anaemic blood on egg production of mosquitoes and found that it has a negative influence on fecundity. Based strictly on egg production and the physics of fluid intake, we propose that although anaemia associated with blood-borne parasites may be detrimental to mosquitoes that can engorge to repletion in one session, it may be beneficial to those interrupted before repletion because the greater quantity of the bloodmeal may compensate for its lower quality. Epidemiological consequences are discussed but require further inquiry.

CBA/T6 and Balb/c mice inoculated with Plasmodium berghei ANKA strain (PbA) died from cerebral malaria 6–8 days post-inoculation. DBA/2J mice similarly inoculated developed a non-fatal cerebral malaria, with mild temporary cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop a cerebral malaria but died between days 15 and 22 from other malaria-related complications. These mouse strain/parasite strain combinations allow for detailed examination of factors critical in the pathology of murine cerebral malaria. Monastral Blue, a colloid dye, when injected intravascularly between days 0 and 2 into PbA-inoculated CBA (PbA-CBA) or Balb/c (PbA-Balb/c) mice prevented death from cerebral malaria. There was no evidence of increased vascular permeability at this stage. When Monastral Blue was injected between days 5 and 8, there was increased vascular permeability in the kidney, liver, lung, spleen and brain of PbA-CBA and PbA-Balb/c mice. Injection of Monastral Blue into these animals at this time also precipitated cerebral symptoms and death, but not in Pb-infected mice. Endothelial and mononuclear cells phagocytosed, and were coated with, the Monastral Blue particles when the dye was injected between days 5 and 8 into PbA-CBA and PbA-Balb/c mice. Control, uninfected mice did not demonstrate either of these features. Pb-infected mice only demonstrated coated mononuclear cells. Mononuclear cell attachment to the endothelium, increased vascular permeability and increased association of Monastral Blue particles with monocytes and endothelial cells were correlated with cerebral symptoms and death. Monastral Blue is thus a useful agent for studying the roles of mononuclear cells and endothelium in murine cerebral malaria.

Babesia hylomysci and B. divergens were studied for superoxide dismutase (SOD) activity by enzyme assay and isoelectric focusing (IEF). In the two Babesia species, parasite-associated SOD is cyanide-insensitive and inhibited by H2 O2, indicating that iron is the cofactor metal. Measurements of SOD activity from purified parasites show that the SOD activity detected in Babesia is, for the main part, due to an endogenous enzyme.

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2·5–25 µl of splenic aspirate or of 50–500 µl of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2·5 × 10−7 µl of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigotelike forms in Schneider's Drosophila medium supplemented with 2% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32–33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5·4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

We describe a new, improved test for the detection of intestinal infection by Entamoeba histolytica. The test depends upon immunoadsorption of the E. histolytica cysteine proteinase, histolysain, from faecal samples, and subsequent visual detection of the enzyme by a colour reaction. With samples from 200 volunteers, results agreed closely with those obtained by the conventional microscopic technique, and there were no false positive reactions with samples containing other parasites. The test is suitable for use either in the laboratory or in the field.

A solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomatic E. histolytica cyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 72·7% of cases of amoebic liver abscess, 18·1% of the cyst passers and 2·5% of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess.

Fractionation of Schistosoma mansoni cercariae gland secretion on a Sephadex G-150 column followed by a Superose-12 column in an FPLC system, isolated a 47 kDa protease which migrated as a single band on SDS–PAGE gels. A monoclonal antibody (MAb) was produced which recognizes only the 47 kDa protease, and an immuno-affinity column with the MAb was used to isolate the protease. The 47 kDa protease showed activity on several macromolecules such as elastin and collagen type VI besides gelatin and casein. This suggests that this enzyme can be one of the enzymes that might facilitate invasion of the cercariae through host skin. The optimal pH of the protease against the synthetic substrate, Ac-Phe-Arg-Nan, in Tris–HCI buffer was 10. Experiments with protease inhibitors indicate that the purified enzyme is a serine protease.

We consider two phenomena, related to the host age-intensity profiles of parasitic infections, which have been suggested to be indicative of acquired immunity: (i) a lower age of peak intensity among more intensely infected hosts; and (ii) a decline with age in the dispersion of the distribution of parasites between hosts. We demonstrate that these phenomena occur among Kenyan schoolchildren infected with Schistosoma mansoni, although the magnitude of both is small. We also examine the mathematical models underlying these predictions and conclude that both phenomena are possible in the absence of acquired immunity or, indeed, in the absence of any density-dependent effect. In our opinion, insufficient attention has been focused upon mathematical models, describing the null hypothesis, i.e. density-independent models. In particular, we regard the usual assumptions made for the two stochastic components of these models, describing the heterogeneity between hosts and the probabilistic nature of infection and death of parasites, as too rigid and unrealistic. We demonstrate that deviation from these assumptions undermines the qualitative distinctions between models which describe acquired immunity or density dependence and those which are density-independent.

The polystomatid Pseudodiplorchis americanus occupies a wider range of habitats and experiences more diverse physiological conditions than any other monogenean. It initially invades the respiratory system of an anuran, Scaphiopus couchii, and then migrates along the alimentary tract while the host is actively feeding, resulting in exposure to digestive enzymes and the surfactant action of bile. Whilst in the urinary bladder, parasites are exposed to wide osmotic fluctuations. This ultrastructural study reveals a set of adaptations unknown elsewhere in platyhelminth biology. During development within the respiratory tract, pre-migratory worms accumulate two types of electron-dense tegumental vesicle. Migration through the gut is accompanied by mass secretion of vesicles which appear to provide protection against digestion. Pre-migrants transferred experimentally to digestive fluids die within minutes unless they have received a specific cue which is responsible for triggering vesicle secretion. Migrants stimulated by this trigger factor can tolerate the same conditions for up to 8 h by continually releasing vesicles to form a protective surface coat. No specific structural adaptations were evident for survival in the lungs or bladder of the host.

Pectoral fins from juvenile rainbow trout (Oncorhynchus mykiss) were used in a bioassay, the object of which was to quantify the effect of simulated shadow stimuli on the transmission success of the cercariae. For a period of 60 min, parasites and fins were exposed to a sequence of computer-controlled shadow stimuli, continuous light or total darkness, and subsequently the number of infections was counted. Trials were conducted with cercariae 1, 12 and 24 h old. Within the constraints of the experimental procedure, shadows were found to increase significantly the number of infections achieved by the 12-h-old parasites. As the cercariae aged, their ability to infect host tissue declined. The relevance of these results to transmission in the natural environment is discussed.

Trickle infections with a hamster-adapted strain of the hookworm Ancylostoma ceylanicum were studied by administering doses of 5–30 larvae twice weekly to inbred DSN hamsters. The worm burdens were regulated at very low levels, and this was found to be independent of the size of the infective dose. It is likely that there is some turnover of adult worms during trickle infection, as larvae of all stages were recovered from most time-points. This regulation is immunologically mediated and both the worm burdens and fecundity were increased in hamsters that were immunosuppressed by cortisone treatment. A trickle infection regime also induced a 67% protective immunity to a single subsequent challenge. The resistance that occurred during trickle infections was, however, incomplete, and some worms were found to survive in hamsters that had been repeatedly infected for over 10 weeks. Thus, although hamsters are capable of regulating the infection, some worms are capable of surviving the host immune effectors.

A simple technique is described for preparation of Giardia cysts from faecal samples, the growth of trophozoites in suckling mice and the isolation of trophozoites for genetic analysis by allozyme electrophoresis. In total, 125 new isolates of Giardia have been collected from human and animal sources over a wide geographical area of South Australia and the Northern Territory of Australia. A number of long-established axenized isolates of G. intestinalis belonging to Groups I and II also adapted to grow in suckling mice. These findings indicate that suckling mice are permissive hosts for a variety of genetically dissimilar but morphologically similar organisms of the G. duodenalis type and that this in vivo technique may be less selective than isolation by in vitro culture. The use of suckling mice has revealed that infections can be composed of mixed genotypes and that isolation and purification techniques can be selective. Allozymic interpretation is essential to reveal the genetic complexity of such mixtures

To facilitate the completion of their life-cycle, many helminth parasites have evolved the ability to manipulate the behaviour of their intermediate host in order to make it more likely to be eaten by the parasite's definitive host. Here, we determined whether the cestode Eubothrium salvelini modifies the behaviour of its intermediate host, the copepod Cyclops vernalis, and makes it more susceptible to predation by brook trout, Salvelinus fontinalis, the parasite's final host. Following the experimental infection of copepods, the spontaneous activity of infected and control subjects was quantified weekly. In addition, we regularly quantified predation by individual brook trout fry on known numbers of infected and control copepods. At approximately the time when the cestode larvae became infective to fish (2–3 weeks following infection), the infected copepods started to swim more actively than uninfected controls. Also at that time, infected individuals became more likely to be captured by fish than uninfected ones. Copepod size and intensity of infection had no significant effect on their behaviour or their risk of being eaten by fish. Thus cestode- induced changes in copepod swimming activity can lead to infected copepods becoming highly vulnerable to fish predators, and may have resulted from selection on the parasite to increase its transmission success

The present study compares parasite-specific antibody responses in two Caribbean communities with high and low levels of Trichuris trichiura transmission. The age-dependency of antibody levels suggest that IgG1 and IgG2 levels relate to the current intensity of infection (as assessed by density of eggs in stool (e.p.g.) and reflect the age–intensity profile at the population level. IgG4, IgE and IgA levels persist into early adulthood and the subsequent decline is gradual. In the low transmission area, lower infection levels are reflected in lower parasite-specific antibody levels (of all isotypes) in the community as a whole. Despite a significantly greater past experience of infection in the high transmission area, antibody levels are not maintained at significantly higher levels throughout adulthood. The production of IgA appears to require a threshold for triggering, and a vigorous IgA response is maintained into early adulthood only in the high transmission village where peak intensity is greatest and the age-convexity of intensity is most marked. Experimental and theoretical studies focusing on the dynamic nature of host–helminth interactions in hosts exposed to high and low infection levels, and the putative role of acquired immunity, are discussed in relation to the data presented.

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.

Toxocara canis infective stage larvae continually produce excretory–secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive post-translational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.

Search behaviour of two entomopathogenic nematode species with different foraging strategies was compared by measuring parameters of unrewarded search after contact with host cues. Steinernema glaseri cruises in search of hosts. Steinernema carpocapsae ambushes hosts. Nematodes should respond to contact with relevant host cues by shifting their search from ranging to localized after contact with them. We predicted that cruising foragers rely on chemical cues more heavily than ambushers. These species were also tested for host affinities. Nematodes were tracked by image analysis after exposure to faeces, cuticle or food of either Popillia japonica or Spodoptera exiqua. Steinernema glaseri responded to selected host cues by shifting from ranging to localized search, characterized by decreased locomotory rate, distance travelled, search area and the proportion of the test period spent moving. Steinernema carpocapsae did not respond to host cues. Steinernema glaseri responds to selected chemical host cues for host location, whereas S. carpocapsae does not.

Genomic DNA extracted from entomopathogenic nematodes isolated from 89 soil samples taken throughout the United Kingdom was hybridized with the ribosomal DNA clone from Caenorhabditis elegans (pCe7). When the DNA was digested with EcoR I and Hind III in a double digest, 5 distinct RFLP (restriction fragment length polymorphism) types were observed. While the prevalence of the 5 types was not equal, no correlation with geographical location, soil type or habitat could be detected. Subsequent hybridizations of total genomic DNA from the various RFLP types divided them into 2 groups. The most prevalent group, identified as Steinernema feltiae ( = bibionis), contained 2 of the RFLP types (Al and A2). The other group contained the remaining 3 RFLP types (B1, B2 and B3). Although similar to S. feltiae ( = bibionis), the members of the B-types can be distinguished from this species on morphological grounds and preliminary crossbreeding experiments have demonstrated that the 2 groups are reproductively isolated.

Proteinase activities were examined in extracts and excretory–secretory (ES) products of the infective and adult stages of the cattle lungworm, Dictyocaulus viviparus. Multiple enzyme activities were identified from each source, as defined by pH optima, substrate specificities, inhibitor effects and substrate gel electrophoresis. Serine-, cysteine- and metalloproteinases were identified, secreted materials being more active against protein substrates per unit protein than were extracts, and the particular proteinases produced varied with the developmental stage of the parasite. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymic activity with Protein G-purified serum IgG antibody from both infected and vaccinated hosts and in the retardation of enzyme migration on electrophoresis of enzyme–antibody complexes. For the adult products, this confirmed that the enzymes concerned were of parasite origin, and not host-derived. These results argue for investigation of D. viviparus proteinases as targets for the antibody response in the limitation of parasite-mediated tissue damage and as the active principle behind the anti-D. viviparus vaccine.