Codon usage has been analysed in individual gene sequences, derived from a variety of parasitic protozoa in the class Sporozoa of the phylum Apicomplexa using metric multidimensional scaling. The two groups of codon usage patterns detected reflect the two main subgroups of organisms studied (the coccidia and the piroplasms), and it is the pattern usage of synonymous codons that has the largest influence on overall codon usage in the individual genes, rather than being the pattern of amino acid composition of the gene product. The magnitude of the codon usage bias in the sequences was determined using three commonly used indices – NC', GC3s and B. In general, although relatively low levels of codon usage bias were detected in these gene sequences, codon usage bias does explain at least some of the codon usage patterns observed. Codon usage bias was observed to be dependent on the overall base composition of the genes analysed, which in turn was reflected in the types of codons that were either over-or under-represented in the nucleotide sequences. keeping with observations on prokaryotic organisms, it is speculated that the codon usage patterns detected in these parasitic protozoa are the result of directional mutation pressure on the base composition of the genomic DNA.

The paper describes a longitudinal field study of canine leishmaniasis in sympatric domestic dog and crab-eating fox (Cerdocyon thous) populations. Dogs were studied in house-to-house surveys, and foxes were studied by live-trapping and radio-telemetry. Because serological tests (IFAT in this case) for leishmaniasis are often of uncertain sensitivity and specificity, we draw conclusions comparatively. Both cross-sectional (age–prevalence) and longitudinal analyses indicate that incidence in dogs was highest in the dry season. Seasonal changes in the age–prevalence relationship for dogs suggest that serological conversion and recovery rates decline with prior exposure to infection, where ‘recovery’ may be due to loss of a positive antibody titre or death from leishmaniasis. The mean incidence in dogs was higher in the rural than in the urban population and higher in hunting dogs than pet dogs. Cross-sectional and longitudinal analyses of fox serological data suggest that foxes remain positive for longer than dogs on average, either because detectable antibody is more persistent or because they experience a lower mortality rate due to leishmaniasis.

Two species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.

Three strains of male and female mice, CFLP, BALB/c and CBA/ca, were infected i.p. with the tetrathyridia of Mesocestoides corti and examined in groups of 5 at 0, 7, 14, 21, 28 and 42 days post-infection. At post-mortem the numbers of parasites both loose in the peritoneal cavity and in the liver tissue were counted, the livers weighed fresh and sections of liver stained to examine the inflammatory response, encapsulation of the tetrathyridia and for eosinophils, neutrophils, mast cells and plasma cells. BALB/c mice had significantly more parasites loose in the peritoneal cavity than CFLP and CBA/ca mice. Infected livers of all three strains were significantly heavier than control livers; the heaviest livers were those of the CFLP followed by the CBA/ca mice which also showed the greatest rate of weight increase. The tetrathyridia from the peritoneal cavities of the CBA/ca and CFLP mice were covered with a ‘mucilage-like’ substance. Tetrathyridia within the host capsules of the CBA/ca mice contained host cells. There was no strain difference with regard to numbers of tetrathyridia in the liver but male mice harboured significantly more parasites than females. Differences in the numbers of cell types within the liver were detected between the strains but no one strain showed any consistent pattern. There was an overall increase in total white blood counts as well as an increase in the number of eosinophils, monocytes, neutrophils and lymphocytes during the course of the infection.

The ability of haemocytes, from the haemolymph of the gastropod mollusc Lymnaea stagnalis, to recognize and eliminate the bacterium Aeromonas salmonicida was shown using an in vitro bacterial clearance assay. The assay employs a dye which is reduced by A. salmonicida in direct proportion to the number of viable bacteria resulting in a colour change which can be determined spectrophotometrically. Addition of cytochalasin B resulted in a marked decrease in bacterial clearance, implicating both intracellular and extracellular cytotoxicity of haemocytes. A comparison of haemocytes from uninfected snails and snails infected with the avian schistosome parasite Trichobilharzia ocellata showed that both juveniles and adults of L. stagnalis were susceptible to infection with T. ocellata. After exposure to the trematode for 1·5 h the haemocytes from these infected snails had an enhanced clearance capacity, whilst cells obtained from snails with 24–96 h infections showed decreased clearance of the bacteria, indicating suppression by the parasite. Haemocytes, as well as plasma, which was tested on haemocytes from uninfected snails, were used and hence a distinction was made between cell and humoralassociated effects. The results show that both cellular and humoral components of immunity were activated, then suppressed, following exposure to the parasite. Infection with T. ocellata seems to have a modulating effect on the bactericidal activity of the internal defence system of the snail host, L. stagnalis.

Field and laboratory investigations into the effect of the parasitic isopod Anilocra pomacentri (Cymothoidae) on the population dynamics of the reef fish Chromis nitida (Pomacentridae) were carried Out at Heron Island, Great Barrier Reef, Australia. Fish carried a single adult parasite just posterior and dorsal to the eye either to the right or to the left of the midline. The adult parasite was overdispersed among fish on patch reefs (dispersion factor, k = 0·69). Sequential field observations on a single cohort of fish showed that parasites significantly depressed growth, reproduction, and survivor-ship. The von Bertalanffy growth coefficients (a measure of somatic growth) were 0·10 for parasitized fish compared with 0·17 for non-parasitized fish. Female fish carrying the parasite produced only 12% of the number of eggs produced by non-parasitized fish of the same size. In the field, the mortality of infected juvenile C. nitida (LCF 15–30 mm) was estimated to be at least 88% in the first 70 days after recruitment of the fish. The mortality of uninfected recruits over the same period was 66%. In laboratory trials, the mortality associated with the infection of juvenile fish by larval parasites ranged from 78% for small fish (mean LCF 15·0 mm) to 28% for larger fish (mean LCF 24·9 mm) within 4 days of experimental infection. This is one of the few studies that evaluates the effect of a parasite on a population of fish in the field.

A protocol for dissociating single muscle fibres from intact flatworms was developed. Muscle fragments of various sizes were obtained, many with their cell bodies, or myocytons, intact. Many of the fibres were spontaneously contractile, and they and others contracted in response to applications of transmitter candidates, activators of protein kinase C and the anthelmintic praziquantel. The responses were all similar to those evoked in strips of tissue. Voltage clamp recordings from the isolated muscle fibres revealed that they possess an inward Ca2+ current and 3 separate K+ currents. These results indicate that muscle fibres in Bdelloura bear receptors for neurotransmitters and that preparations of dispersed muscle fibres can be used for studying the basic physiological and pharmacological properties of platyhelminth muscle.

We have previously described the major yolk protein, vitellin, in the British blackfly Simulium ornatum Meigen. Here we demonstrate that vitellogenin, synthesized in the fat body and secreted into the haemolymph, is composed of subunits with the same approximate molecular weight as vitellin, namely 200 and 68 kDa. Simulium ornatum is the natural vector for the cattle filarial nematode Onchocerca lienalis Stiles, which induces host fecundity depletion. A significant reduction in ovarian vitellin content was associated with infection by intrathoracic injection of 20 O. lienalis microfilariae immediately after blood-feeding. Fat body synthesis of vitellogenin was significantly reduced as early as 8 h post-infection in comparison with sham-injected flies. When total haemolymph protein from infected and sham injected flies was compared, titres were significantly depressed 6 h post-infection. However, later in the infection, titres were elevated by 30%, the major component being vitellogenin. The injection of dead microfilariae had no effect. An infection burden of a single parasite caused a significant reduction in ovarian protein content in comparison with shams, but no further significant decrease was observed as the parasite burden was increased from 5 to 20. Possible mechanisms underlying the disturbance of Simulium reproductive physiology are proposed.

A novel repetitive DNA sequence in the sheep parasitic nematode Trichostrongylus colubriformis was cloned and sequenced. A l·1 kb repetitive sequence (Tc15) which hybridized with DNA from T. colubriformis but not with DNA from two other parasitic nematodes, Haemonchus contortus and Ostertagia circumcincta, or sheep was further characterized. Southern blot analysis showed that the repeat hybridized to a range of fragments in restriction digested T. colubriformis DNA and existed in multiple copy number tandem arrays. However, to define clearly the repetitive monomeric unit further screening of phagemid libraries containing BamH I restriction fragments using a subclone of Tc15 as a probe was carried out. Restriction map and sequence data were compiled for 3 clones containing a 145 bp highly repetitive sequence (designated TcREP) which shared homology with the original pTc15 clone. TcREP hybridized to a tandemly repeating sequence monomer of 145 bp in T. colubriformis DNA which was cloned from various genetic environments in the T. colubriformis genome. TcREP homologous sequences were also found in the genomes of two other species of the same genus (Trichostrongylus axei and Trichostrongylus vitrinus) but not in a fourth species (Trichostrongylus rugatus).

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C- terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.

Sensitive assays capable of detecting proteinases in single females of the phytoparasite Globodera pallida have been developed and used to define the proteinase activity of young adult females. Digestion of the large subunit of the plant protein Rubisco established a pH optimum for the proteinase activity at pH 5·7. The activity was inhibited by the cysteine proteinase inhibitors p-chloromercuribenzoic acid (PMBA) and p-chloromercurisulphonic acid (PMSA) and stimulated by both cysteine and dithiothreitol (DTT). It was moderately reduced by L-trans-epoxysuccinyl-leucylamido-(4- guanidino) butane (E64) but not by specific inhibitors of serine, aspartate or metallo-proteinases. The activity separated into 3 bands on a non-denaturing gel but only I proteinase of 62 kDa was recovered following a combination of anion-exchange chromatography and affinity chromatography using PMBA. The effect of inhibitors was similar to that reported previously for some of the cysteine proteinase activity recovered from Caenorhabditis elegans but is apparently not that for which the corresponding gene has been cloned in this nematode and Haemonchus contortus. The proteinase may have a major role in digestion of dietary protein and so offers an exciting target for future control of this important plant-parasitic nematode.

Wetanema sp. is a nematode parasite of the hind gut of the freezing-tolerant orthopteran Hemideina maori (a New Zealand alpine weta). The prevalence and intensity of infection remains high throughout the winter, suggesting that the parasite can survive despite the regular freezing of the host. In the laboratory, Wetanema has survived freezing within its host to temperatures as low as −61 °C, much lower than the supercooling point or lower lethal temperature of the weta. The freezing tolerance of the parasite is therefore much greater than that of its host. Female worms survived lower temperatures better than males and juveniles. Parasites might assist the freezing tolerance of their host if they acted as endogenous ice nucleators. However, there was no relationship between the size of the worm burden and the supercooling point of the host and no significant difference between the supercooling points of infected and uninfected hosts. The freezing and subsequent survival of isolated Wetanema was observed directly on a microscope cold stage. This parasite of a freezing- tolerant host is thus also freezing tolerant. There are few other reports of a parasite surviving freezing within a living host.

This paper presents a method of estimating the potential global morbidity due to human intestinal nematode infections (Ascaris lumbricoides, Trichuris trichiura and hookworms), based on the observed prevalence of infection. The method relies on the observed relationships between prevalence and intensity of infection, and between worm burden and potential morbidity. This approach is shown to be sensitive to the precision of the original prevalence estimates and, in particular, to the degree of spatial heterogeneity in levels of infection. The estimates presented here indicate that several tens of millions of children are likely to suffer developmental consequences from infection, and suggest that the global disease burden of geohelminthiasis may be significantly greater than was supposed.

Epidemiological modelling can be a useful tool for the evaluation of parasite control strategies. An age-structured epidemiological model of intestinal helminth dynamics is developed. This model includes the explicit representation of changing worm distributions between hosts as a result of treatment, and estimates the morbidity due to heavy infections. The model is used to evaluate the effectiveness of different programmes of age-targeted community chemotherapy in reducing the amount of morbidity due to helminth infection. The magnitude of age-related heterogeneities is found to be very important in determining the results of age-targeted treatment programmes. The model was verified using field data from control programmes for Ascaris lumbricoides and Trichuris trichiura, and was found to provide accurate predictions of prevalence and mean intensities of infection during and following different control regimes.