The effects of dietary protein and zinc restrictions on Heligmosomoides polygyrus were compared following primary and challenge infection in female BALB/c mice fed either control (24%), marginal (7%) or low (3%) protein combined with either high or low zinc (60 or 3 mg Zn/kg diet). Dietary protein restriction (3%) resulted in significantly lower body weight gain. As well, blood urea nitrogen (BUN) significantly decreased with decreasing dietary protein level. However, neither plasma albumin concentration nor relative thymus or spleen weights were reduced. Marginal zinc deficiency was confirmed by significantly lower tibia and liver zinc concentration, but food intake, body weight gain, relative thymus and spleen weights, and alkaline phosphatase activity were not altered. On day 29 post-primary infection, worm burdens were significantly higher in mice fed either marginal or low protein and in mice fed a low zinc diet, while parasite egg output was significantly higher in mice fed both low protein and low zinc diets. Immune status was compromised in mice fed low protein (significantly lower serum IgG1 and lower eosinophilia), and in mice fed low zinc diet (significantly lower eosinophilia). Early in the infection, IgE titres were elevated in mice fed low protein or low zinc, but IgE titres declined to levels lower than the control diet groups after 14–21 days. On day 29 post-challenge infection, worm burdens and parasite egg output were significantly higher in mice fed low protein, whereas the other groups had expelled almost all parasites. Dietary restriction had no effect on serum IgE. Significantly reduced serum IgG1 titres and eosinophilia in mice fed 3% protein supported the view that low dietary protein but not low zinc increased host susceptibility to H. polygyrus by compromising host immune function following reinfection in immunized mice.

The effect of digestion in trypsin and acid pepsin on Toxoplasma gondii tachyzoites and bradyzoites was re-evaluated because of recent use of this method to distinguish tachyzoites from bradyzoites. Toxoplasma gondii tachyzoites survived better in 0·5% trypsin solution for 1 h than in 1·0% solution, and occasionally survived for 2 h in acid pepsin solution. Extracellular tachyzoites ([ges ]1000) were also infectious orally to mice and cats. Bradyzoites survived equally in trypsin and acid pepsin solutions but the digestion of brain tissue in 0·5% trypsin solution was better than in acid pepsin solution. The resistance to digestion in acid pepsin solution is not a reliable method to distinguish tachyzoites from bradyzoites.

The uptake of Wuchereria bancrofti microfilariae (Mf) by Culex quinquefasciatus and their development in relation to human Mf density were quantified by allowing a total of 1096 wild mosquitoes to feed on 13 volunteers sleeping under partially open bed-nets. For each volunteer, each hour between 18.00 and 06.00 h the Mf density in finger-prick blood was determined and engorged mosquitoes collected. Each hourly collection of mosquitoes was kept separately. Half of them was dissected within 18 h post-feeding for the presence of ingested Mf, the other half was reared for 12 days to allow for the development of L3 larvae. About 20% of the latter mosquitoes died during these 12 days and these harboured significantly more larvae than the surviving ones, which could be an indication of excess-mortality among heavily infected mosquitoes. Assuming that variability in Mf uptake and in the number of developed L3 larvae can be described by a negative binomial distribution, a maximum-likelihood procedure was applied to estimate the relationship between human Mf density and both the arithmetic mean Mf uptake and L3 development. Both were adequately described by a saturating hyperbolic function that significantly differed from linearity. The saturation level for Mf was estimated at 29 (CI: 20–54) and for L3 larvae at 6·6 (CI: 4·3–17·0). Next, the L3 yield was related to Mf uptake indicating that the W. bancrofti–C. quinquefasciatus complex shows ‘limitation’, i.e. a decreasing yield for an increasing uptake. Both the number of Mf ingested and the number of L3 larvae developing per mosquito were found to be highly aggregated, with the level of aggregation decreasing in a non-linear way with human Mf density.

We have used a styryl dye FM 1–43 that cannot cross membranes passively to evaluate membrane vesicle internalization processes from the heptalaminate surface membrane in schistosomula and adult Schistosoma mansoni. The process of dye internalization was faster in adult worms than in 3 h skin or 24-h-old mechanically transformed schistosomula. Dye internalization and vesicle formation were inhibited at low temperature which suggests that the process is energy dependent. Dye internalization was observed to be a result of vesicle formation. This process is impaired by primaquine independently of the parasite stage. The specific Ca2+ chelator EGTA affected dye internalization at a concentration of 5 mm. When parasites were labelled in the presence of genistein (a tyrosine kinase inhibitor) uptake of the dye was blocked in 24-h-old schistosomula. However, only a partial blockage of the dye internalization was seen in adult worms. These results suggest that dye internalization mediated by tyrosine kinase signalling is developmentally regulated.

The polymerase chain reaction (PCR) method was used to characterize trypanosome infections in tsetse flies from 3 sleeping sickness foci in Cameroon. The predominant tsetse species found was Glossina palpalis palpalis. An average infection rate of 12·1% was revealed by microscopical examination of 888 non-teneral tsetse flies. PCR amplification analyses for trypanosome identification were carried out on 467 flies, with primer sets specific for Trypanosoma (Trypanozoon) brucei s.l., T. (Duttonella) vivax, T. (Nannomonas) simiae and forest type T. (Nannomonas) congolense. Of 467 flies 93 were positive by microscopical analysis while PCR succeeded in identifying 89 positive flies. Of the PCR-positive flies 34 (38·2%) were negative by microscopical examination. PCR amplification, when compared to the parasitological technique, gave a higher estimate of infection rate of trypanosomes in natural tsetse populations. The PCR technique did, however, fail to identify 40·9% (38/93) of the parasitologically positive flies. The reasons for this failure are discussed. The overall prevalence of mixed infections, assessed by PCR, was 37·1%; the majority (72·7%) involved T. brucei and forest type T. congolense.

In North Cameroon, the vector of Onchocerca volvulus (causative agent of human onchocerciasis) also transmits 2 filariae of animals: O. ochengi from cattle and O. ramachandrini from wart hogs. In order to assess the qualitative and quantitative roles of these ‘animal filariae’ in the epidemiology of O. volvulus, the transmission of the 3 parasites was measured in 2 villages and related to the endemicity of human onchocerciasis. In Galim, a cattle-farming Guinea savanna village where wild animals are rare, the overwhelming majority of all filarial infections found in the Simulium damnosum s.l. vectors throughout the year were O. ochengi (89%). The remaining infections were mainly O. volvulus (10·5%), and a few O. ramachandrini (0·5%). In Karna, a crop-farming Sudan savanna village where cattle are rare, but wild animals common, flies were also more frequently infected with animal filariae than with the human parasite. In the dry season, when nomadic cattle are present, 54% of all infections were O. ochengi, 36% O. volvulus and 10% O. ramachindrini. In the rainy season, when the cattle move away, flies were mainly infected with O. ramachandrini (52% of all infections) and secondly with O. volvulus (48%). In Karna, the relationship between the Annual Transmission Potential (ATP) of O. volvulus and its prevalence in the human population conformed to other onchocerciasis foci, in that a moderate ATP led to hyperendemic onchocerciasis. In Galim, however, a 7-fold higher O. volvulus-ATP (caused by a very high biting rate of the flies) contrasted with a strikingly low endemicity of onchocerciasis. Since, at the same time, in Galim the transmission of O. ochengi (measured on man) was very high (15000 L3/fly collector/year), we hypothesize that the reduced endemicity of onchocerciasis in Galim is due to ‘natural heterologous vaccination’ by the large annual number of O. ochengi-L3, inoculated into man by anthropo-boophilic S. damnosum s.l. The importance of micro-epidemiology for the understanding of the interlinkage of human and animal onchocerciasis is discussed.

Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria. The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed. Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane. Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent. We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle. We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts. The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size. This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17·5S, containing the 9S rRNA, and 20S containing the 12S rRNA. A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2[ratio ]3 ratio (20S[ratio ]17·5S).

Several recent studies have discussed the interaction of host life-history traits and parasite life cycles. It has been observed that the life-history of a host often changes after infection by a parasite. In some cases, changes of host life-history traits reduce the costs of parasitism and can be interpreted as a form of resistance against the parasite. In other cases, changes of host life-history traits increase the parasite's transmission and can be interpreted as manipulation by the parasite. Alternatively, changes of host's life-history traits can also induce responses in the parasite's life cycle traits. After a brief review of recent studies, we treat in more detail the interaction between the microsporidian parasite Edhazardia aedis and its host, the mosquito Aedes aegypti. We consider the interactions between the host's life-history and parasite's life cycle that help shape the evolutionary ecology of their relationship. In particular, these interactions determine whether the parasite is benign and transmits vertically or is virulent and transmits horizontally.

Key words: host-parasite interaction, life-history, life cycle, coevolution.

The kinetics of serum antibody response against infection with Echinococcus multilocularis eggs was evaluated in AKR mice. The animals were infected by oral inoculation with 300 parasite eggs, and necropsied at 1, 2, 4, 6, 9, 12 and 16 weeks post-infection (p.i.), respectively. The parasite formed the laminated layer at 4 weeks p.i., the brood capsule with a massive proliferation of germinal cells at 9 weeks p.i. and protoscoleces at 16 weeks p.i. Serum antibody responses of the mice to antigen preparations from metacestodes of different stages and protoscoleces were evaluated by ELISA, immunoblotting and immunohistochemistry. In ELISA, the antibody responses began to increase at 4 weeks and became more apparent at 9 weeks p.i. and thereafter. Immunoblots using sera collected at 16 weeks p.i. showed some common bands among the 3 different antigen preparations. In addition to this, the germinal cells and brood capsules of mature metacestodes were stained strongly in an immunohistochemical study. From above, it is suggested that some antigen molecules are expressed in the parasite through these stages and stimulated host antibody responses.

Onchocercomata with a defined worm population were analysed to elucidate the distribution of mast cells. Nodules with live females were classified according to the presence or absence of microfilariae. Immunohistochemical staining was performed using antibodies specific for mast cells or IgE. Mast cells appeared singly or in diffuse accumulations perivascularly and in inflammatory infiltrates between adult Onchocerca volvulus and in the capsular area. No mast cells were detected in cystic parts. Only few, scattered mast cells were found in the fibrous zone around the adult worm. They were increased with stronger infiltration and hence, related to the inflammatory cells. Mast cells were never localized directly at adult worms or microfilariae. A correlation of the mast cell distribution to the occurrence of eosinophils was observed regarding higher numbers of mast cells and eosinophils in nodules with microfilariae-producing females. Nodules with single males revealed higher numbers of mast cells than nodules with non-producing females, although both contained very few eosinophils. Onchocercomata with dead worms contained significantly more mast cells than those with live filariae. In conclusion, the localization and frequency of mast cells is contingent on the vitality and productivity of the worms and therefore, indirectly and directly on the release of O. volvulus antigens.

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.

Parasitoids are parasites that invariably kill their host. Polydnaviruses are injected by parasitoid wasps into the body cavity of their insect host and cause immunosuppression, allowing the parasitoid to develop in the absence of encapsulation. One of the targets of the polydnaviruses are the haemocytes of the host, which undergo significant changes in response to entry of the virus. In some systems, haemocyte apoptosis is induced, or haemocyte clumping may be seen; in others, the cells round up and fail to adhere to a substrate. Effects on haemocytes may be transitory or permanent (cell death). Various polydnavirus gene products have been identified that interfere with normal haemocyte function. Phenoloxidase activity also is inhibited during parasitism, and the effect is inducible by polydnavirus. In some systems, venom components may act synergistically with polydnavirus in mediating the virally-induced effects on the host immune system. Polydnaviruses are powerful influences on the host immune system, which serve to permit successful development of the parasitoid without triggering the host immune response.

Electrophysiological and pharmacological techniques were used to study glutamatergic signalling in the parasitic nematode, Ascaris suum. Glutamate or kainate injections into whole worms produced a paralysed quasi-static posture similar to the waveform in behaving worms. The DE2 motorneuron class is a primary target. Several glutamatergic substances produced pronounced conductance increases and depolarization in DE2; domoate and kainate were the most potent agonists tested. Glutamate responses and spontaneous excitatory post-synaptic potentials in DE2 were reversibly blocked in sodium-free saline. DE2 sensitivity to exogenous glutamate was sustained during block of synaptic transmission suggesting that glutamatergic receptors are located on DE2 neurons. The glutamate-induced response was localized to the DE2 dendrite, coincident with the synapses responsible for spontaneous potentials in DE2. Steady-state potentials reached during glutamate superfusion were similar to the reversal potentials for both the spontaneous post-synaptic potentials and glutamate, also suggesting that these potentials may be glutamatergic. Non-N-methyl-D-aspartate receptor antagonists partially blocked spontaneous DE2 excitatory potentials and responses elicited by exogenous glutamate and kainate. This glutamatergic pathway may play a role in nematode locomotory behaviour and account for the paralysing anthelmintic action of excitatory amino acid analogues like kainate and domoate.

A benznidazole-resistant population of Trypanosoma cruzi, Y strain, was selected after 25 successive passages (8 months) in mice treated with a single high drug dose. Initially, the resistant parasites produced a low parasitaemia level and low mortality rate in infected mice. Thereafter, the parasitaemia level and mortality rate increased to the same value obtained for mice infected with the wild-type strain. Long-term treatment with benznidazole (100 mg/kg/day) cured 71–80% of mice infected with the wild-type strain. No cure was observed in mice infected with the selected resistant parasite population. Treatment with 500 mg/kg of benznidazole at peak parasitaemia cleared all blood parasites from mice infected with wild-type parasites. No effect on parasitaemia level was observed in mice infected with the selected parasites. Benznidazole-resistant parasites showed cross-resistance to different drugs. Contrary to wild type, all clones analysed from the resistant T. cruzi population were resistant to benznidazole. Without drug pressure the resistance phenotype of clones was far more stable than that presented by the resistant population. This work demonstrates, for the first time, the in vivo selection of a population and clones of T. cruzi resistant to benznidazole, and makes available an experimental model for the study of mechanisms of drug resistance in T. cruzi.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to α granules or β granules of T. spiralis, but no resemblance to γ granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E–S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction led to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS–PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

Reciprocal interactions of parasites transmitted by blood-sucking arthropod vectors have been studied primarily at the parasite–host and parasite–vector interface. The third component of this parasite triangle, the vector–host interface, has been largely ignored. Now there is growing realization that reciprocal interactions between arthropod vectors and their vertebrate hosts play a pivotal role in the survival of arthropod-borne viruses, bacteria, and protozoa. The vector–host interface is the site where the haematophagous arthropod feeds. To obtain a blood meal, the vector must overcome the host's inflammatory, haemostatic, and immune responses. This problem is greatest for ixodid ticks which may imbibe as much as 15 ml blood whilst continuously attached to their host for 10 days or more. To feed successfully, the interface between tick and host becomes a battle between the host's mechanisms for combating the tick and the tick's armoury of bioactive proteins and other chemicals which it secretes, via saliva, into the feeding lesion formed in the host's skin. Parasites entering this battlefield encounter a privileged site in their vertebrate host that has been profoundly modified by the pharmacological activities of their vector's saliva. For example, ticks suppress natural killer cells and interferons, both of which have potent antiviral activities. Not surprisingly, vector-borne parasites exploit the immunomodulated feeding site to promote their transmission and infection. Certain tick-borne viruses are so successful at this that they are transmitted from one infected tick, through the vertebrate host to a co-feeding uninfected tick, without a detectable viraemia (virus circulating in the host's blood), and with no untoward effect on the host. When such viruses do have an adverse effect on the host, they may impede their vectors' feeding. Thus important interactions between ticks and tick-borne parasites are displaced to the interface with their vertebrate host - the skin site of blood-feeding and infection.

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.

A cross-sectional study of bancroftian filariasis was conducted in 2 adjacent communities, Mvumoni and Kilore in Muhaka, Kwale District. Wuchereria bancrofti infection, clinical manifestations and anti-filarial IgG responsiveness were determined before the long rains, a time coinciding with a low transmission season. The prevalence of microfilaraemia increased gradually with age and was significantly higher in Kilore (24%) than in Mvumoni (6·3%, P<0·001). Similarly, the prevalence of antigenaemia increased with age and also was significantly higher in Kilore, 48·9% than in Mvumoni, 20·5% (P<0·001). Hydrocele, funiculitis, lymphangitis and lymphadenitis were also significantly more common in Kilore than in Mvumoni. In comparing the 2 communities, levels of IgG4 responsiveness in antigen-positive persons were higher in Kilore than Mvumoni (P=0·034), but this was related to higher antigen loads in persons in Kilore than in Mvumoni. In antigen-negative persons, anti-filarial antibodies of 3 IgG isotypes were significantly higher in Kilore than Mvumoni (P<0·001, for IgG1, IgG2, IgG4). These results emphasize the highly focal nature of bancroftian filariasis in this setting and demonstrate that anti-filarial antibody levels are related to transmission intensity.

Electrophysiological and pharmacological experiments suggest the presence of an electrogenic glutamate transporter in the motornervous system of the parasitic nematode Ascaris suum. This putative transporter occurs in hypodermis (a tissue in some respects analogous to glia) and in DE2 motorneurons, a dorsal excitatory motorneuron class which receives excitatory glutamatergic post-synaptic potentials. Glutamate application to hypodermis produced non-conductance mediated depolarizations that were smaller in amplitude and slower in rate of rise than DE2 responses where a glutamate-activated conductance occurs. The hypodermal response is sodium dependent and calcium independent. Excitatory amino acid ionotropic receptor agonists (kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid and N-methyl-D-aspartate) were ineffective in eliciting hypodermal responses. The ionotropic receptor antagonist, 6,7-dinitroquinoline-2,3-dione, had no effect on hypodermal glutamate responses. The L- and D-forms of glutamate, aspartate and homocysteate produced hypodermal and DE2 depolarizations consistent with the pharmacological profile for glutamate transporters in other systems. Glutamate transport inhibitors (L-trans-pyrrolidine-2,4-dicarboxylate and beta-hydroxyaspartate) elicited electrogenic depolarizations in hypodermis and DE2. These results suggest that the hypodermal glutamate response has an electrogenic transporter component, while the DE2 response has 2 components, one conductance-mediated and the other due to an electrogenic transporter.