DAMP (3-(2,4-dinitroanilino)-3′amino-N-methyldipropylamine), which differentially accumulates in acidic compartments, was used to identify such compartments in Toxoplasma gondii tachyzoites at the electron microscope level. In both free tachyzoites and dividing intracellular parasites the only sites of DAMP accumulation were mature and forming rhoptries. No labelling of other secretory organelles (micronemes and dense granules), the ER, Golgi or any other membrane-bounded organelles or anything resembling a lysosomal system was observed. Labelling of the forming rhoptries was higher and more homogenous than in mature rhoptries in which labelling was confined to the expanded ends of each organelle. The acid pH-dependent accumulation of DAMP in the forming and mature rhoptries was blocked by ammonium chloride and monensin, reagents known to abolish intracellular pH gradients. Estimates of rhoptry pH, based on the level of DAMP accumulation, show that the intralumenal pH of forming rhoptries is more acidic (pH 5·5–3·5) than the mature rhoptries (pH 7·0–5·0).

Gyrodactylus salaris, an important pathogen of Atlantic salmon Salmo salar, has been shown to be highly sensitive to factors in host serum and mucus, being killed rapidly (50% within 1h) by serum at a dilution of 1[ratio ]200. The time needed for killing was inversely proportional to serum concentration. Similar effects were noted using host mucus, which contained approximately 1/20th of the anti-Gyrodactylus activity of serum. Serum activity was abolished completely by heating at 45°C for 30 min, and by addition of EDTA, but not by EGTA+1 mm magnesium ions. Activity was not dependent on whether the serum was from infected or naive fishes, nor was it species specific. Attempts to pre-coat parasites in salmon anti-Gyrodactylus antibodies also failed to enhance the activity of fresh serum. These observations suggest that killing is due to the complement system of the host, acting via the alternate pathway. G. salaris appears to be exceptionally sensitive to complement, being killed at concentrations which could be experienced in vivo. The role of complement in the protection of fishes against gyrodactylid infection therefore deserves further investigation.

In order to investigate the molecular taxonomy within Trypanosoma cruzi, the ribosomal small subunit (18S) gene was amplified by polymerase chain reaction (PCR) from a selection of 21 stocks and 3 outgroup taxa. Amplification products were digested with 10 restriction enzymes; restriction fragments were separated by polyacrylamide gel electrophoresis and profiles were visualized by silver staining. Upon analysis of such riboprint profiles, an estimate of pairwise phenetic distance between stocks of T. cruzi was calculated. Upon principal coordinate analysis of this data matrix, a tendency towards a bi-polar grouping of stocks was observed. These 2 groups were predominantly either zymodeme 1 stocks or zymodeme 2 stocks. The position of zymodeme 3 stocks remained intermediate between the 2 groups but did not form a coherent group by themselves. It would therefore appear premature to warrant division of T. cruzi into 2 discrete taxa or subspecies until the relationships of further zymodeme 3 stocks are elucidated.

A gene (vsp417-3A-II) encoding a new member of the variant-specific surface protein (VSP) family of Giardia is described. Vsp417-3A-II is detected exclusively in isolates belonging to the genetic Assemblage A/Group II sublineage of G. intestinalis, and it is closely related to a previously characterized VSP gene (tsp11, herein renamed vsp417-2A-I) found in Group I isolates of G. intestinalis. Analysis of DNA amplified from the genomes of Group II isolates using consensus primers also revealed products corresponding to the vsp417-2 locus (vsp417-2A-II allele), indicating that vsp417-2A-II and vsp417-3A-II represent separate loci in these organisms. Southern hybridizations revealed a single genomic copy of vsp417-3A-II in Group II isolates, but efforts to detect this locus in Group I organisms were unsuccessful. Together with the vsp417-1 (tsa417) locus that was first identified in the genetic Group I isolate WB and which has been detected since in all tested genetic Group I isolates (as vsp417-1A-I) and in genetic Group II isolates (as vsp417-1A-II), vsp417-3A-II represents a third member of a stable subset of tsa417-like VSP genes in genetic Group II G. intestinalis. The encoded polypeptide, vsp417-3A-II (667 residues, predicted Mr 69120) possesses a high cysteine content (12·1 mol%), 28 copies of the metal-binding -CXXC- motif, and a highly conserved C-terminal hydrophobic domain – similar to all other characterized Giardia VSP. A revised nomenclature for Giardia VSP genes is proposed, prompted by the need to describe the homologues found in the various major genotypes of the species complex, G. intestinalis.

There is an essential requirement for highly sensitive tools that will differentiate nematode parasites of animals and plants to the species level. For studying host range, genetic variation, virulence and resistance, the availability of well defined populations is vital. Many nematode species cannot be identified with certainty using traditional morphological or morphometric techniques. This is particularly the case for the more accessible developmental stages that, depending on the particular group concerned, live as eggs and larvae in the environment or as micro-filariae that circulate in the blood or inhabit the skin. Morphological identification of these stages requires specialized expertise and is extremely time consuming. Immunological assays have their place in nematode identification but they do not discriminate between current and previous infections, an essential requirement in many epidemiological and prevalence studies. In addition to being highly sensitive, DNA-based methods of detection define present over past infection and are not dependent on the parasite stage. Many types of methodology are available for the detection and definition of nematode DNA. This paper reviews these methods citing examples that have been used with success in the laboratory as well as the field.

The present study examines antigenic variability for the human whipworm Trichuris trichiura. Recognition by IgG of somatic antigens of individual worms collected from 3 intensely infected children from Jamaica, West Indies has been investigated by immunoblotting. When probed with 1 plasma sample, significant differences in recognition of 2 selected antigens among worm populations and between male and female worms was observed. In addition, there was evidence for antigenic variability within worm populations at the individual worm level. Such variation may have considerable implications for the development of immunity to parasitic nematodes.

The levels of superoxide dismutase (SOD) were determined in detergent-soluble, somatic and excretion–secretion (E–S) preparations from adult Fasciola hepatica using the xanthine oxidase system and visualized in substrate gels. Compared to detergent-soluble and somatic extracts, E–S products showed the highest SOD activity (88 ·5 U/mg), indicating active release to the medium in which parasites were maintained. SOD specific activity was also detected at high levels in E–S products from 3-week-old and 5-week-old immature migrating flukes (25 and 143 U/mg, respectively). In all preparations except for the somatic extract, the activity was characterized as cyanide-sensitive CuZn SOD. Differences in SOD isoenzyme profiles between the extracts were observed in native polyacrylamide gel electrophoresis: the somatic and detergent-soluble extracts exhibited 1 band of activity while the E–S products from immature and adults flukes contained 2 and 3 migrating bands, respectively. SOD was purified from the detergent-soluble extract and E–S products of adult worms by a combination of ultrafiltration, gel filtration on Sephacryl S-200 HR and ion-exchange chromatography on QAE Sephadex A-50. The SOD from detergent-soluble extract showed, by SDS–PAGE analysis, 1 band of 16 kDa apparent molecular weight. The SOD from E–S products showed 2 bands of 16 and 60 kDa apparent molecular weight. N-terminal sequence analysis of the 16 kDa band from the detergent-soluble preparation showed some similarity with Schistosoma mansoni cytoplasmic SOD. These enzymes may have a potential role in the evasion of the oxidative burst killing mechanism by immune cells.

The genetic differences between praziquantel-resistant (R) and susceptible (S) strains of Schistosoma mansoni (Fallon & Doenhoff, 1994) were explored using RAPD and by cloning differentially expressed mRNAs by subtractive PCR. No differences between the 2 strains were detectable by RAPD using 41 different primers indicating that no major genomic rearrangements were present. Subtractive PCR generated a number of fragments, 1 of which was shown to correspond to an over-expressed mRNA in the R strain and to encode a fragment of the subunit 1 of cytochrome-c oxidase (SCOX1). In the absence of a complete sequence for this gene, we used EST sequences to compile a consensus sequence for the 904 bp at the 3′ end that enabled us to choose primers for semi-quantitative RT–PCR. This technique showed that SCOX1 was indeed over-expressed about 5 to 10-fold in the R strain whereas the genes encoding the 28 kDa glutathione S-transferase, glutathione peroxidase, NADH dehydrogenase subunit 5 and the ATP-binding cassette family protein SMDR2 were not. In contrast, cytochrome-c oxidase enzyme activity was 4-fold lower in the R strain than in the S strain.

Recent work has shown that mice with subclinical parasitic infections suffer impaired spatial learning and memory, as assayed in an open-field water maze. Although the mechanism underlying this effect is not clear, the phenomenon has been reported following infection with both a protozoan parasite (Eimeria vermiformis) and a gastrointestinal nematode (Heligmosomoides polygyrus). In a variety of experiments, we examined the effects of a different gastrointestinal nematode, Strongyloides ratti, on the ability of rats and mice to learn a spatial or a discrimination task. Animals were tested at various stages post-infection, with different levels of infection, using different lines of S. ratti and with varying experimental protocols. All animals learned the tasks, but we found no evidence of an effect of S. ratti infection on learning or memory. Even rats infected with approximately 5000 S. ratti larvae, a dose which has an impact on rat body size, showed no deficit in learning ability. Various reasons for the conflict between our results and those previously reported for E. vermiformis and H. polygyrus are discussed. Our results show that impaired learning and memory following parasitic infection is not a ubiquitous or at least easily replicated phenomenon.

It has recently been shown using genetic markers that Ascaris in humans and pigs in Central America comprise reproductively isolated populations. We present a similar analysis for a region of China in which close association between pigs and humans has been the norm for thousands of years, and agricultural practices will result in frequent exposure to eggs from both sources. DNA fragments from selected regions of mitochondrial and ribosomal DNA were amplified by PCR and allelic forms identified following digestion with a panel of restriction enzymes, using DNA from a total of 115 individual worms from both people and pigs from 2 neighbouring villages. Significant frequency differences in both mtDNA haplotypes and the rDNA spacer were found between the 2 host-associated populations, indicating that they represented reproductively isolated populations. Mitochondrial haplotype frequencies were different from those observed in Guatemala and also from other Asian Ascaris populations, suggesting low levels of gene flow between populations. However, we found no evidence for significant heterogeneity in the genetic composition of Ascaris infrapopulations in either humans or pigs, possibly indicative of agricultural practices in China which have resulted in a random distribution of alleles within the parasite populations.

The ascaridoid nematode of cats from Kuala Lumpur, Malaysia, previously identified morphologically as Toxocara canis, was characterized using a molecular approach. The nuclear ribosomal DNA (rDNA) region spanning the first internal transcribed spacer (ITS-1), the 5·8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. The sequences for the parasite from Malaysian cats were compared with those for T. canis and T. cati. The sequence data showed that this taxon was genetically more similar to T. cati than to T. canis in the ITS-1, 5·8S and ITS-2. Differences in the ITS-1 and ITS-2 sequences between the taxa (9·4–26·1%) were markedly higher than variation between samples within T. canis and T. cati (0–2·9%). The sequence data demonstrate that the parasite from Malaysian cats is neither T. canis nor T. cati and indicate that it is a distinct species. Based on these data, PCR-linked restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) methods were employed for the unequivocal differentiation of the Toxocara variant from T. canis and T. cati. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and zoonotic potential of this parasite.

Adults of the monogenean genus Protopolystoma infecting Xenopus species occur in an extremely space-limited habitat, the urinary bladder. Xenopus wittei, from a population in Rwanda naturally infected with Protopolystoma fissilis and Protopolystoma simplicis, were exposed to reinfection in captivity (for 1–3 months post-capture) and then monitored in the laboratory for up to 5 months in transmission-free conditions. The two parasites co-occurred in individual bladders less frequently than expected if they were dispersed randomly. Distribution of bladder infections was significantly non-independent (n=157) and gravid worms of both species were never found in the same host. This pattern might be explained by interference competition between the parasites or by genetic differences in susceptibility within the host species, which is of allopolyploid origin. Other distributional data for sympatric polystomatid species pairs, including P. fissilis and P. ramulosus, show concurrent infections at frequencies consistent with random distributions (i.e. no evidence of interspecific competition or variability in species-specific susceptibility of the hosts). Interference between P. fissilis and P. simplicis (assuming host genetic factors are not involved) may therefore result from a mechanism specific to this species pair. Observations on infection turnover in captive hosts suggest that loss of adult worms may be related to the arrival of juveniles (of either species) in the urinary bladder. Ectopic infection of the host urinary ducts by adult and subadult P. fissilis was observed in some single-species infestations and may be density related. However, the use of an ectopic-site ‘refugium’ has never been observed in concurrent polystomatid infections.

We describe a laboratory model using Eimeria falciformis and E. pragensis to investigate some of the interactions in double-species infections of Eimeria. Mice were given trickle infections by oral inoculation of 100 sporulated oocysts of one species at 3 or 4 day intervals throughout the experiments and, once immunity had developed, as indicated by cessation of oocyst production, the animals were challenged with a single inoculation of the other species. A trickle infection of E. falciformis gave a significant enhancement of oocyst output from E. pragensis infection as compared with animals that had not received E. falciformis. Histological examination of the mice infected with E. pragensis showed significantly more asexual parasites and a significantly higher female/male ratio in the mice that had received trickle E. falciformis infections than in those that had not. There was no evidence to suggest that extra asexual generations were occurring in these mice. In the converse experimental protocol, trickle E. pragensis infections had no significant effect on a single E. falciformis infection. We discuss the possible mechanisms for the interactions and also how these interactions may influence multiple species infections in animals in their natural habitats.

The importance of foodborne parasitic zoonoses remains high in many regions of the world. Although control efforts have been exerted for quite some time, overall progress has not been satisfactory, even in many well developed countries. An important drawback in control programmes for parasites such as Trichinella, Toxoplasma and Taenia has been the absence of rapid, accurate and sensitive diagnostic tests for these meatborne parasites. However, the rapid advances in the molecular biology of these organisms has yielded concomitant gains in precision of detection. This review highlights these advances and their impact or potential application to the control of foodborne parasites.

To test the homogeneity of 18S sequences within Trypanosoma cruzi, riboprint profiles were separated by temperature gradient gel electrophoresis (TGGE). Upon interpretation of melting curves of fragments within a riboprint profile, there appeared to be two 18S sequence types within each stock examined. Two similar types were also observed within outgroup taxa Trypanosoma conorhini, Trypanosoma rangeli and Leishmania braziliensis. From DNA hybridization studies, these fragments were shown to have homology to the 18S V1 region. There are therefore two 18S V1 regions, differing in sequence, present in all taxa examined. When only a single 18S sequence is used to represent each taxa for phylogenetic inference, comparisons may be between paralogous and not orthologous copies of this region, such that, inferred relationships may merely reflect a gene history. This seriously questions the current molecular phylogeny of these protozoa using 18S data.

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

The importance of the third (L3), fourth (L4 and adult stages of Mecistocirrus digitatus in inducing resistance to reinfection was examined. Three groups of 5 calves (A, B, C) were immunized for 2 consecutive days with a daily dose of 20000 M. digitatus infectious larvae. Group A was treated with ivermectin 7 days post-infection (exposed to L3), group B was treated after 26 days (exposed to L3and L4) and group C after 60 days (exposed to L3, L4 and adults). Thirty days post-treatment, animals were challenged with 30000 M. digitatus L3. Five previously uninfected control animals (group D) also received 30000 L3. All animals were necropsied 75 days post-challenge. Increases in exposure to the immunizing infection resulted in significant increases in the pre-patent period post-challenge; 54 days in the controls, compared with 63, 70 and 72 days for groups A, B and C, respectively. Only adult worms were recovered at necropsy, and the mean number of worms significantly increased with increasing exposure to the immunizing infection: 355 in the controls, compared to 481, 937 and 1174 in groups A, B and C, respectively. No significant changes in worm length were observed. Infection with M. digitatus significantly affects a subsequent challenge infection – stimulation of the immune system by exposure to L3 extends the pre-patent period, and suppression by later stages (L4, adult) leads to higher worm burdens.

The impact of mixed, nematode infection upon a group of animals will depend upon the number of nematodes present, how they are distributed among hosts and whether individuals that are heavily parasitized with one species are more likely to be heavily parasitized with other species. A survey of over 500 six-month-old, Scottish Blackface lambs from a single farm in Southwest Strathclyde identified 7 different categories of nematodes in the abomasum and small intestine. There were considerable differences among years and among nematodes in the prevalence and mean intensity of infection. Ostertagia circumcincta was present in nearly all lambs and judged by prevalence and intensity is one of the most successful of all parasitic nematodes. Each category of nematodes had a skewed distribution; most animals had relatively few worms but a small proportion had many worms. The variances of the number of nematodes in each category were approximately equal to the square of the mean. The counts of adult O. circumcincta followed a negative binomial distribution, but the negative binomial distribution did not provide a good description of the observed values for the other species. These other species had a lower prevalence and possibly some sheep were not exposed to infection. There was no significant genetic variation among lambs in the number of nematodes present and therefore the differences among these lambs were unlikely to be a consequence of genetic differences in host susceptibility. Lambs with increased numbers of one species were more likely to have increased numbers of the other species, but the correlations were weak and may reflect covariation in exposure to different parasites.

The initiation of further development is fundamental to the infectious processes of parasitic nematodes. We have examined early developmental activation of Trichinella spiralis larvae during host invasion, with particular emphasis on the timing of events. Using a novel approach, we have observed changes in tissue-specific transcriptional activity in live larvae during the infectious process with the fluorescent nucleic acid dyes SYTO12 and acridine orange. Simultaneously, the metabolic switch from anaerobic metabolism, characteristic of the infective stage, to aerobic metabolism, as found in the enteral stages, was tracked by measuring activities of the key regulatory enzymes phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as isocitrate dehydrogenase (NADP) activity, and used as a co-indicator for developmental activation. Both metabolic enzyme activities and transcription patterns were found to change in response to host death, liberation from the nurse cell, and exposure to components of the host stomach environment. The results give a clear indication that the activation processes of T. spiralis infective larvae occur at a much earlier time than previously thought, and are stimulated upon liberation of the larvae from the nurse cell inside the host stomach.

The circumstances which permit the establishment of Leishmania infections in sandflies were investigated by altering the growth conditions for L. donovani parasites in the unsuitable vector Phlebotomus papatasi. Only 5·0% of the sandflies harboured a few parasites 3 days after feeding on promastigotes in defibrinated blood. Heparinized blood or the addition of trypsin inhibitor to the meals allowed persistence of infections (day 6) in 9·9% and 25·8% of the flies respectively. Meals of erythrocytes, saline and amastigotes produced 44·4% fly infection on day 6, while similar promastigote-initiated infections remained in 70·3% of the flies. Proteolytic activities in the guts of sandflies fed on the above meals without parasites, were the highest after defibrinated bloodmeals. Erythrocytes with saline decreased the maximal alkaline protease level from 20·8 U to 13·5 U/fly; that of trypsin from 3·9 U to 1·8 U/fly and that of the aminopeptidase from 5·5 U to 3·9 U/fly. After meals of heparinized blood, the maximal alkaline protease activity (12·0 U/fly) was also much lower than after defibrinated blood-feeding. The different diets which resulted in comparatively low enzymatic activities, including blood with trypsin inhibitor, also promoted the survival of infections. This implies that the proteolytic activity in the sandfly gut modulates the vector susceptibility.