The combination of pyrimethamine and sulfadoxine (PSD or Fansidar) represents one of the most important chemo-therapeutic agents currently used to treat falciparum malaria. To investigate the molecular basis of resistance to PSD, reliable in vitro drug assays are required to permit correlation of resistance levels with different genotypes. We describe here protocols that permit accurate evaluation of IC50 values for sulfadoxine (SDX) inhibition of Plasmodium falciparum. Historically, tests for this drug have suffered from poor reproducibility and extreme variability in reported values. We have examined a series of variables, including serum-containing versus serum-free media, erythrocyte source, pre-test growth conditions, test components and post-test processing. We define conditions which better control the levels of the drug antagonists folate and p-aminobenzoate, yielding reproducible differences between lines of P. falciparum with differing alleles of the dihydropteroate synthetase gene, which encodes the target enzyme of SDX. We also use this assay to demonstrate a marked difference in the response of different parasite lines to antagonism of SDX inhibition by exogenous folate. The ability to measure reliable IC50 values for SDX inhibition should greatly facilitate further experiments to explore the molecular basis of Fansidar resistance.

Multiplication of virulent and avirulent Leishmania donovani promastigotes was inhibited by brain heart infusion broth (BHI) (3·7%) supplemented with haemolysed or whole blood (2·5–10%) or heat-inactivated foetal bovine serum (HIFBS) (10–20%). Peptone, glutamine, tryptophan, purine bases, ribonucleosides, deoxyribonucleosides, B-vitamins and vitamin C were evaluated for the reversal of this growth inhibition. Supplementation of BHI with folic acid further supplemented with haemolysed rabbit blood or HIFBS and haemin supported propagation in serial subpassages of 2 primary isolates and 3 other virulent strains of L. donovani and 1 strain of L. major. Folic acid supplemented BHI with 3% rabbit blood in solid phase with 0·7% agar also supported the colony formation of L. donovani. This manuscript documents the development of high efficiency media for the cultivation of L. donovani promastigotes in liquid phase and as colonies on agar plates using BHI as a basal medium.

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-dl-phenylalanine β-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and evidence obtained after partial purification, SDS–PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody ‘responder’ mice were significantly (P<0·001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

The potential of a recombinant Schistosoma bovis-derived glutathione S-transferase (rSb28GST) to protect cattle against S. mattheei infection was tested in Zambia. All animals were challenged 2 weeks after the second inoculation with either 0·250 mg rSb28GST in adjuvants (vaccinated calves, n=14) or adjuvants alone (controls, n=14). In a first experiment, 7 vaccinated and 7 control animals were exposed to 10000 S. mattheei cercariae percutaneously. All animals developed clinical schistosomiasis 7–8 weeks after challenge. At perfusion, 12 weeks after challenge, vaccinated and control groups had averages of 887 and 541 eggs per gramme (epg) faeces, 6515 and 5990 worms, and 4·2 and 3·4 million tissue eggs, respectively. These results indicate that the immunization protocol used did not protect these calves against the massive single experimental challenge. In a second experiment, another 2 groups (n=7) of vaccinated and control animals were challenged naturally over a period of 9 months on a farm known to be endemic for S. mattheei. The natural infections were much lighter in intensity, as indicated by the mean faecal egg count (13 epg), worm count (139) and tissue egg count (294000) in non-vaccinated controls. In vaccinated calves, significant reductions in female worm burdens (50%), faecal egg counts (89%) and miracidial counts (93%) were recorded. Total tissue egg counts were also reduced by 42% in vaccinated animals. It therefore appears that the rSb28GST can provide significant protection in cattle against S. mattheei under conditions of low to moderate natural infection.

The aim of the study was to measure the impact of Schistosoma japonicum and Trichuris suis infections in young growing pigs fed low- or high-protein diets. Thirty-two pigs, 6–10 weeks old, were randomly allocated to 2 groups receiving either a high- or a low-protein diet. After 11 weeks half of the pigs from each group were infected with 1500 S. japonicum cercariae and 4000 T. suis eggs. The weight of the pigs was measured throughout the study, and blood and faecal samples were collected every second week from the time of infection. At the time of infection the low-protein pigs had significantly lower mean body weights, haemoglobin and albumin levels compared with the high-protein pigs, and this pattern continued throughout the study. The serum albumin concentration was further significantly reduced in the infected low-protein pigs compared to the non-infected low-protein pigs. Significantly more S. japonicum worms as well as faecal and tissue eggs were found in the low-protein pigs compared with the high-protein pigs. No differences between the 2 diet groups were observed in T. suis establishment rates or faecal egg excretion. We conclude that this low-protein diet increased the establishment rates of S. japonicum, favoured larger deposits of S. japonicum eggs in the liver and faecal egg excretion, reduced weight gains and caused anaemia and hypoalbuminaemia in younggrowing pigs as compared with a high-protein diet.

We present a simulation population model for the African tick Rhipicephalus appendiculatus, based on previous analyses of the mortality factors most closely correlated with observed population changes at 11 sites in equatorial and South Africa. The model incorporates temperature-dependent rates of egg production and development, climate-driven density-independent mortality rates, particularly during the adult-larval stage, and density-dependent regulation of both nymphs and adults. Diapause is also included for tick populations in southern Africa. The model successfully describes both the seasonality and annual range of variation in numbers of each tick stage observed at each of 4 test sites in Uganda, Burundi and South Africa. Sensitivity analysis showed that the final version of the model is robust to 4-fold variation in most parameter values (that were per force based on informed guesses), but is more sensitive to the regression coefficients determining density-dependent interstadial mortality (that were derived from analysis of field data). The model is able to predict the seasonality of ticks from a site in Kenya where a full prior population analysis was not possible because only adults and nymphs had been counted. The model is potentially applicable to other species of ticks, both tropical and temperate, to predict tick abundance and seasonality as risk factors for tick-borne diseases.

The actions of known platyhelminth FaRPs on the contractility of whole-worm preparations of the monogenean, Diclidophora merlangi have been examined in vitro for the first time. All of the peptides tested had excitatory effects on the motor activity of the worm. The order of potency for the peptides tested was: YIRFamide > GYIRFamide = RYIRFamide > GNFFRFamide = FLRFamide. However, although YIRFamide was more potent than GYIRFamide, the latter was the most efficacious on each of the motility parameters (tension, contraction amplitude and contraction frequency) examined at concentrations [ges ]0·1 μm. Serotonin, which stimulates contractility in the worm was used as a positive control. The excitatory activity of turbellarian and cestode neuropeptides on a monogenean indicates at least some structural similarities in the neuropeptide receptors of these classes of flatworm.

The neuromuscular sodium currents of early invertebrates such as platyhelminths display distinctive kinetic and pharmacological properties. We have cloned a cDNA from the horseshoe crab flatworm Bdelloura candida that encodes a protein homologous to the primary subunit of voltage-gated sodium channels. The B. candida protein, named BdNa1, exhibits amino acid identity of 40–47% to sodium channels of vertebrates and higher invertebrates. BdNa1 has the multidomain structure characteristic of sodium channels, and is most highly conserved in the hydrophobic transmembrane segments and the regions that form the pore of the channel. Northern blot analysis confirms the presence of a 5·4 kb BdNa1 transcript in B. candida tissue. The information provided by analysis of the BdNa1 sequence offers insight into the physiology of platyhelminth sodium currents.

Interactions between adult Hymenolepis diminuta and rat C-reactive protein (CRP) were investigated in vivo and in vitro. Using an ELISA technique, serum levels of CRP were monitored in rats infected with 100 cysticercoids. Although infection increased the level of this protein in the early stages of parasitization, the increase was not significant until 35 days post-infection (p.i.). Secondary infections did not enhance the response. When H. diminuta was cultured in the presence of CRP, reduced worm motility and opaque areas were observed and electron microscopical studies revealed shedding of microtriches and lysis of the tegument. Initially, damage was restricted to the strobila which correlated with the regional distribution of phosphorylcholine as visualized using immunofluorescence.

The influence of nutrient level, temperature and larval density on the trapping of Heligmosomoides polygyrus L3 by the nematophagous fungi Arthrobotrys oligospora, Duddingtonia flagrans and Monacrosporium megalosporum were investigated by quantification of trapped nematodes. All 3 factors were found to have a significant effect on the number of larvae trapped by A. oligospora and M. megalosporum. Decreased nutrient concentrations resulted in increased trapping for these 2 fungi, but nutrient availability was not found to have a significant effect on trapping by D. flagrans. The 3 fungi were found to have similar responses to temperature, with peak trapping occurring at or near the optimum growth temperatures. Nematode trapping was found to be density dependent for all 3 fungi, with increased percentage trapping at increased larval densities. Comparison in a single experiment of the relative importance of these factors to each fungus showed that nutrient level was the main factor influencing trapping by A. oligospora, whereas D. flagrans and M. megalosporum were more dependent on larval density.

Electrophysiological techniques and behavioural assays were used to determine the response of males of Globodera rostochiensis to 4 fractions of the homospecific sex pheromone from virgin females, separated using reverse-phase high-pressure liquid chromatography. Sex pheromone activity was confined to 2 of the fractions. The sex pheromone of G. rostochiensis appears to be composed of several weakly basic, polar compounds.

In an experiment to study the infectivity, growth and virulence of Schistocephalus solidus in their first intermediate host, copepods of the species Macrocyclops albidus were kept singly and exposed to up to 9 coracidia. Eleven or 14 days post-infection (p.i.) the presence and growth of the cestode larvae relative to survival, growth and reproduction of their host was determined. As expected, the probability of a copepod becoming infected increased with increasing numbers of parasites administered. However, the chances of a single coracidium establishing in a copepod also increased with increasing numbers of coracidia administered, which indicates that the parasites profit from a dilution effect of the host's defence. Copepod size or developmental stage had no significant effect on the infection, but 14 days p.i., constraining effects of copepod size on the growth of the parasites were apparent. Moreover, procercoids in multiple infections grew smaller and developed their cercomer at a smaller size than those in single infections. No significant effect of the parasite on host mortality was found within the observation period. However, growth between the 5th copepodid stage and adult stage was negatively affected by infection. An infection with S. solidus was also strongly linked with host reproduction: infected females were more likely to bear an egg sac at the end of the experiment than non-infected ones. These egg sacs, however, contained fewer eggs.

We describe a variety of restriction site polymorphisms in the introns of Ascaris nuclear genes and in the ribosomal DNA spacers. We use these markers, in addition to previously described mitochondrial variation, to clarify our understanding of the epidemiology of Ascaris in Guatemalan villages where humans and pigs occur in sympatry and to describe the genetic structure of host-associated Ascaris populations from world-wide locations. Intron sequences were amplified from individual worms and alleles defined by endonuclease digestion. Two loci were monomorphic, while 4 length variants and 22 point mutations were detected in the other 7 loci. Within sympatric Guatemalan populations no single locus from either the nuclear or mitochondrial genome was fixed for alternative alleles, although allele frequencies were significantly different at many loci. Phenograms constructed from multilocus nuclear genotypes of individual worms failed to reveal a single case of cross-infection, and demonstrate that divergent mtDNA genotypes are segregating within host-associated populations. On a world-wide scale, the data suggest that extant worm populations result from a single host shift, although characterization of genetic variation in additional loci will be necessary to confirm this. The direction and the geographical origin of the host shift were unresolved. Overall 65% of nuclear genetic variation was found within populations, host (human or pig) explained 18%, while geographical variation within host-associated populations explained 17%. The results (a) demonstrate the utility of introns for studying the epidemiology of parasites showing limited allozyme variation (b) suggest that programmes aiming to control Ascaris infection in the human population can safely ignore zoonotic infection from pigs and (c) illustrate the problems inherent in using single genetic markers to make inferences about the epidemiology of closely related parasite taxa.