The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness (with the ability to survive in moist microclimates for prolonged periods of time) pose persistent threats to public and veterinary health. Increased demands made on natural resources increase the likelihood of encountering environments and produce contaminated with parasites. In the last 30 years, endemic and epidemic waterborne and foodborne outbreaks in developed countries have led to a reappraisal of conventional isolation and detection methods. While these methods have proved invaluable in our understanding of environmental transmission routes for helminths, they have been less effective for the parasitic protozoa. Robust, efficient detection, viability and typing methods are required to assess risk and to further epidemiological understanding. Greater awareness of parasite contamination of our environment and its impact on health has precipitated the development of better detection methods. Currently, nowhere is this more apparent than with Cryptosporidium, with a broad range of immunological, microscopical and molecular methods available. The upsurge in molecular techniques, particularly the polymerase chain reaction, for determining occurrence and viability have brought with them the added benefits of increased sensitivity and specificity, yet many methods still have to be shown to address these issues consistently in the field. Rapid commercialization of reagents and standardization of methods provide consistency. The advances identified in non-destructive and destructive methods for the protozoa have application for helminths and emerging pathogens and should determine the importance of the matrices involved in the environmental transmission of parasites, further safeguarding public and veterinary health.

The maturation and quantification of Theileria lestoquardi (T. hirci) parasites in unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was studied using (1) methyl green pyronin (MGP) staining of salivary glands, (2) in vitro infection of peripheral blood mononuclear cells (PBM) with parasites harvested from infected ticks and (3) a semi-quantitative polymerase chain reaction (PCR). With MGP staining the greatest infection rate was seen in unfed ticks. Feeding resulted in a gradual reduction in the number of infected acini with a concomitant increase in the maturity of the parasites. In vitro infection of sheep PBM with titrated ground-up tick supernate (GUTS) demonstrated that infectivity peaked between 2 and 4 days of tick feeding whereas GUTS prepared from unfed ticks was not infective. The polymerase chain reaction (PCR) was both sensitive and specific, detecting T. lestoquardi DNA in unfed and partially fed ticks, with a maximum sensitivity of 0·022 infected acinus/tick in 2-day fed ticks, though it gave no indication of the infectivity of the parasite.

We compared, some fitness-related traits of benzimidazole resistant (rr) and susceptible (rS, SS) worms of Teladorsagia circumcincta, a gastrointestinal parasite of the small ruminants, under laboratory conditions. PCR was used to determine the genotypes (rr, SS, rS) and the fitness of each was compared within the same strain. There was no significant difference in egg production, development rate from egg to infective larvae stage, establishment of these larvae in the host or the survival of adult worms and infective larvae for the 3 genotypes. The same results were obtained for the establishment rate of larvae in the host and the production of infective larvae under conditions of strong competition between resistant and susceptible worms. The fact that there were no differences in fitness suggests that the installation of benzimidazole resistance in a worm population is irreversible. This agrees with field observations.

A rabbit antiserum was raised against a synthetic peptide corresponding to a region near the N-terminus of the Haemonchus contortus inhibitory amino acid receptor subunit, HG1. The antiserum recognized a recombinant form of the N-terminal domain of the subunit on Western blots and reacted with the ventral nerve cord of H. contortus in immunofluorescence experiments. Immunofluorescence was also detected in specific head neurons of H. contortus: these were tentatively identified as ring motor- and inter-neurons, plus a possible sensory neuron equivalent to the AQR cell of Caenorhabditis elegans. In the roundworm Ascaris suum, immunoreactivity was limited to the muscle arms, the post-synaptic component of the neuromuscular junction. The possible ligand of receptors containing the HG1 subunit is discussed in the light of this expression pattern.

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers d-glucose and d-mannose but not by l-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

We review the theoretical framework for exploring the impact of individual and spatial heterogeneities in patterns of exposure and contamination and on the basic reproduction number, R0, for human schistosomes. Analysis of water contact data for 5 communities in Zimbabwe and Mali suggests that the impact is substantial, increasing R0 by factors of up to 6·5, mostly due to highly overdispersed distributions of contact rates among individuals. Several practical conclusions emerge: concentration of contacts at a single site should be avoided; the impact of control targeted at certain sites cannot be predicted without knowledge of how individuals' contacts are distributed among sites; control programmes targeted at individuals or sites contributing most to transmission can be very efficient but, conversely, will be ineffective if any of these individuals or sites are missed.

A 120 kDa antigen produced by juvenile female Litomosoides sigmodontis (Juv-p120) was isolated and purified. The amino acid composition of the molecule was determined. Juv-p120 was shown to be highly modified with N,N-dimethyl-aminoethanol (28·4 mol%). Treatment of Juv-p120 with potassium hydroxide (β-elimination) or with sodium m-periodate leads to the destruction of epitopes recognized by antibodies immune affinity-purified with isolated Juv-p120. Juvenile L. sigmodontis were shown to release Juv-p120 into the pleural cavity of infected Mastomys coucha before the onset of patency.

The cestode Schistocephalus solidus is a simultaneous hermaphrodite that grows in 2 intermediate hosts and reproduces rapidly within a few days in the gut of a bird. Reproduction takes place by self- or cross-fertilization. Here, it was tested whether egg production differs between S. solidus that reproduce alone and those that are allowed to reproduce in pairs. Egg production in an in vitro system was found to depend on the cestodes' social situation. When kept alone, larger cestodes produced larger eggs. This was not so when kept in pairs – the difference between these 2 reproductive modes being highly significant in this respect. Further experiments revealed that, within the first 3 days, these hermaphrodites produced a larger total egg mass when kept alone than when kept in pairs. This was also reflected by the energy contents of the cestodes after this time-span: selfers had used up more energy than paired worms. Furthermore, S. solidus appeared to adjust its investment per egg depending on whether the offspring will be the result of self- or cross-fertilization. Selfers produced larger numbers of eggs, but these eggs were smaller and contained even smaller embryos per given egg size than eggs of potentially outbreeding cestodes. Selfed eggs reached lower hatching rates. Although this is to be expected from inbreeding depression it may also be an effect of the reduced maternal investment per egg. The observed phenotypic plasticity in the reproduction of S. solidus is discussed within 4 evolutionary frameworks: local mate competition adjusted for hermaphrodites, the hermaphrodite's dilemma, bet-hedging, and sib-competition.

Isospora belli, Cyclospora cayetanensis as well as several species of microsporidia are recognized as emerging protozoan pathogens of humans. All are obligate intracellular parasites, with Isospora and the microsporidia being primarily associated with immunocompromised hosts. Cyclospora is a cause of traveller's diarrhoea, and is responsible for water-borne and food-borne outbreaks of disease. Drug treatment is available for these infections. Improved diagnostic methods including the autofluorescence of I. belli and C. cayetanensis oocysts have assisted in the routine detection of these pathogens. Since the recognition of immunosuppression due to HIV infection, microsporidia have become recognized as important human pathogens with a continuing expansion of the parasite-associated clinico-pathological spectrum. The small size, intracellular nature and poor staining properties with many histological stains result in under-reporting of microsporidial infections. Trichrome stain and optical brighteners are used to detect spores in faeces, urines, respiratory secretions and other aspirates. Electron microscopy remains an important diagnostic method but its sensitivity is relatively poor. Molecular techniques should overcome current diagnostic limitations. The ability to extract DNA and amplify by PCR directly from clinical samples has increased the usefulness of molecular methods. Restriction fragment length polymorphism analysis of amplicons can be used to determine genus, species and strain types of various microsporidia. Increased specificity is required in primer design because current primers used for amplifying non-microsporidian DNA also amplify microsporidian DNA. Diagnosis and pathogen characterisation rely increasingly on PCR-based approaches, and the sequence analysis approach becomes increasingly feasible and affordable. However, robust, reliable and sensitive methods are still required for dissecting pathogenesis, epidemiology, transmission routes and sources of infections.

Diagnostic PCR primers for Trichinella were constructed. Twelve pairs of primers were designed based on the sequences of random amplified polymorphic DNA, and 4 pairs of primers were designed based on the reported sequences of complementary DNA encoding excretory–secretory glycoproteins. With these primers, 31 samples of DNA from different strains of Trichinella including 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic level (Trichinella T5, T6 and T8) were tested with PCR. Genus Trichinella can be identified by 4 different primer pairs (SB147D, SB372A, SB153, or Ts43). Trichinella spiralis can be identified by the presence of a 673 bp amplicon in PCR with the primer pair SB147B. Trichinella nelsoni can be identified using primer pair SB147F or by the presence of 673 bp and ca. 380 bp amplicon in PCR with the primer pair SB147B. Trichinella pseudospiralis can be identified by 2 primer pairs (SB147E or SB372B). Trichinella T5 can be identified by the primer pair SB147G. Trichinella T8 can be identified by its positivity by the primer pair SB147C and its negativity by the primer pair SB372C. A group of Trichinella species (T. britovi, T. nativa and Trichinella T6) can be identified by the primer pair SB372C.

In this chapter, the contribution of molecular tools in understanding the aetiology and ecology of infectious diseases is examined in the context of molecular epidemiology (ME). ME is seen as providing the ‘tools’, both laboratory and analytical, which have predictive significance in epidemiological investigations of the causation of disease. A diversity of questions can be addressed with these tools which can conveniently be viewed as particular regions of DNA and grouped according to the different hierarchical levels of specificity by which infectious agents can be characterized. These groupings and the applications of the different molecular tools are described, and consideration given to the most appropriate methods of analysing data from ME investigations.

This paper describes the distribution of Ascaris suum in experimentally and naturally infected pigs, and offers a comparison with A. lumbricoides infections in humans. In the first study, worms were recovered post-mortem from a group of 38 pigs that had been trickle inoculated with 10000 infective A. suum eggs twice weekly for 12 weeks. In the second study, worms were collected from a group of 49 pigs that had been kept on a pasture contaminated with infective A. suum eggs for 10 weeks, after which they received treatment with an anthelmintic; they then were turned out on the same pasture for a second 10-week period before slaughter. The worm burdens of the naturally infected pigs were recorded both at treatment and post-mortem. Mean worm counts were similar at all occasions but the prevalence of infection was higher in the trickle infected and naturally reinfected pigs. Furthermore, the prevalence in naturally infected pigs increased significantly over the study period. Worm burden distributions in all groups were heavily overdispersed, but the distribution patterns differed significantly between groups: lower exposure (initial natural infection) gave a low prevalence and an almost uniform distribution of worm burdens among infected hosts. Continued or higher exposure (trickle and natural reinfection) resulted in increased prevalence and a reduction in the proportion of hosts with increasing worm load. A positive correlation was found between initial and reinfection worm burdens in the naturally infected pig population, suggesting that individual pigs are predisposed to a high or low intensity of infection. The prevalence and intensity as well as the distribution observed for A. suum infection in pigs were comparable to those reported for A. lumbricoides in endemic areas, and there is evidence for predisposition to A. suum in pigs, with an estimated correlation coefficient similar to that found in humans. It is concluded that A. suum infections in pigs are a suitable model to study the population dynamics of A. lumbricoides in human populations.

Species of Echinococcus (Cestoda: Taeniidae) require 2 mammalian hosts to complete their life-cycle; a carnivorous definitive host, and a herbivorous or omnivorous intermediate host. For most species of Echinococcus, the definitive host range is restricted to 1 or a few species, but the intermediate host range is very broad. Programmes to control hydatid disease attempt to break the life-cycle of the parasite and their effectiveness is therefore enhanced by an understanding of local patterns of transmission. Although it is known that the rostellar hooks of protoscoleces may be influenced by the species of intermediate host in which they develop, the application of this knowledge to infer transmission cycles has been limited, because the intermediate host effect has not been isolated from other environmental and genetic components of phenotypic variance. This study presents a method for separating these potentially confounding genetic and environmental effects, by combining quantitative genetic analyses of hook traits with data on population structure from neutral genetic markers. The method was applied to 5 hook traits (hook number, total length of large hooks, blade length of large hooks, total length of small hooks, blade length of small hooks) measured on protoscoleces from 2 intermediate host types (sheep and macropod marsupials) in Australia. Although genetic variance was similar for all traits, they differed markedly in the extent of environmental variance attributed to development in different host types. Total length of small hooks was the trait most affected, with 49–60% of phenotypic variance being explained by environmental differences between intermediate host species. Blade length of small hooks was least affected, with none of the phenotypic variance due to intermediate host origin. These data suggest that hook measurements of adult worms from naturally infected definitive hosts could be used to determine the intermediate host species from which infection was acquired, if the appropriate traits are measured.

The macroparasites Cephenemyia trompe (Modeer) and Hypoderma (=Oedemagena) tarandi (L.) (Diptera: Oestridae), Linguatula arctica Riley, Haugerud and Nilssen (Pentastomida: Linguatulidae), Elaphostrongylus rangiferi Mitskevich (Nematoda: Protostrongylidae), and abomasal nematodes (Nematoda: Trichostrongylidae) were sampled in semidomestic reindeer calves (Rangifer tarandus (L.)) (ca. 8 months of age) in northern Norway in 1988 (n=160) and 1989 (n=191). Each parasite showed an aggregated (clumped) distribution among the hosts and fitted the negative binomial distribution. Analyses of interspecific associations in intensities showed that there was no consistent covariation among the parasites apart from a weak correlation (Kendall's tau 0·104, P=0·007) between the 2 oestrids C. trompe and H. tarandi. This lack of covariation reveals that the parasites were distributed independently of each other, and suggests that innate host resistance is not a dominant factor that has a significant simultaneous effect on all parasites. The aggregated distribution of each parasite species is hypothesized to be caused by (1) random events and heterogeneities in host behaviour that create unequal transmission (exposure) rates, or (2) by heterogeneities in parasite specific immunocompetence among host individuals. Factors in hypothesis (1) are probably the most important at low transmission rates.

We compared a Trypanosoma cruzi clone unable to infect or induce pathology in mice (CL-14), with virulent T. cruzi (Y and CL strains) in terms of cruzipain expression, subcellular distribution and functional activity. Our results showed that (1) intracellular Y amastigotes expressed R1 (carboxy-terminal) and R2 (catalytic) domains concentrated in cytoplasmic vesicles, while CL-14 presented R1 labelling on membrane clusters and R2 in intracellular compartments, (2) CL-14-trypomastigotes presented R1 and R2 staining preferentially on flagellar and cellular membranes, similar to CL, but different from Y strain intracellular labelling pattern, (3) flow-cytometry revealed higher expression of R1 by CL-14-trypomastigotes than virulent strains, but R2 staining similar to CL-trypomastigotes, (4) CL-14-trypomastigotes presented normal cruzipain activity in gelatin gels, but different banding patterns were found in CL-14 versus CL and Y strains. Our data rule out failure in cruzipain expression, activity or subcellular distribution as an explanation for CL-14 biological behaviour, but suggest the expression of a different isoform. These results also cast doubt on the putative role of cruzipain as a target of immunopathological responses, since high levels of functional cruzipain are expressed by a non-pathogenic T. cruzi.

Traditionally, the family Campulidae has been associated either with the family Fasciolidae, parasites of ruminants, or the Acanthocolpidae, parasites of fishes, based on morphological similarities. Since morphology does not seem to resolve clearly the problem of the relationships of campulids, we have used the sequences of the 18S rRNA gene of the campulids Zalophotrema hepaticum, Campula oblonga and Nasitrema globicephalae, the fasciolid Fasciola hepatica, the acanthocolpid Stephanostomum baccatum and the outgroup Schistosoma mansoni to infer a phylogeny. Maximum parsimony and neighbour-joining methods were applied. Both methods indicated that campulids are closer to acanthocolpids than fasciolids. In order to confirm this relationship, we generated a second phylogeny using all the partial sequences of the 18S published for trematodes: Lobatostoma manteri, Echinostoma caproni, Calicophoron calicophorum, Tetracerasta blepta, Gyliauchen sp. and Opistorchis viverrini, plus those mentioned above, and Dicrocoelium dendriticum. The aspidogastrean L. manteri was used as the outgroup. Results were identical to the first analysis. According to this and the most recent Digenean phylogeny, which considers campulids and acanthocolpids as sister groups, we suggest that a common origin for these 2 groups would imply a host-switching process. The life-cycle of acanthocolpids includes marine gastropods as first intermediate hosts, and fishes as second intermediate and definitive hosts. In this context, the hypothesis would be that trematodes whose cycle ended in fishes were able to switch to mammalian hosts.

The epidemiology of the sucking lice Echinophthirius horridus was studied over a 4 year period in which their harbour seal hosts experienced marked inter-annual changes in food availability. Prevalence and intensity of infection varied in relation to host age but not sex. Burdens were highest on immature seals, but both prevalence and intensity of infection were significantly higher in years when food availability was low. Observed intra-population variations in ectoparasite dynamics suggested that reported geographical differences in the prevalence of E. horridus may have resulted from methodological differences. In contrast to previous studies of other pinnipeds, lice were also absent on weaned pups, suggesting that E. horridus is transferred horizontally. No significant differences were found in the haematological parameters of infected and uninfected hosts. Nevertheless, there was a significant negative correlation between intensity of infection and several erythrocyte parameters, suggesting that high burdens of lice may compromise diving ability.

Using microelectrode structures, various forms of electric fields, such as non-uniform, rotating and travelling wave, can be imposed on particles of sizes ranging from proteins and viruses to micro-organisms and cells. Each type of particle responds to the forces exerted on them in a unique way, allowing for their controlled and selective manipulation as well as their characterization. Moreover, particles of the same type but of different viability can be distinguished in a simple, reliable manner. This review outlines the principles that govern the way in which bioparticles respond to these various field types, and how they can be exploited. Examples of current and potential biotechnological and biomedical applications are given, along with a critical comparison of current techniques.

A new category of signalling molecules – transmitter gases – has appeared. Nitric oxide (NO) is generated by nitric oxide synthase (NOS) and diffuses as a short-lived transcellular messenger through the plasma membrane. NADPH-diaphorase (NADPH-d) is a marker enzyme for NO-producing neurons. In this study the pattern of NADPH-d stained neurons in Diphyllobothrium dendriticum is described and compared to the pattern of aminergic and peptidergic neuronal elements and to that of the musculature stained with TRITC-labelled phalloidin. NADPH-d staining was observed in neurons in the bilobed brain and along the 2 main nerve cords and in nerve fibres close to the body musculature and the musculature of the reproductive ducts, the walls of the testicular follicles and in sensory endings in the tegument. The NADPH-d staining reaction and the 5-HT or FMRFamide immunoreactivities occur in separate sets of neurons.

The study was conducted to evaluate a mAb-based coproantigen detection assay for monitoring fox prevalence of Echinococcus multilocularis infections in the Shiretoko National Park in Hokkaido, Japan. Eight fox families, each consisting of 2–4 adults and their pups, were selected because their territories have been determined. Faeces of each family (total n=537) were collected monthly from April to October, 1994. Detection of coproantigen and taeniid eggs was recognized in 58 and 27 faeces, respectively, but mostly restricted in 1 family, in which coproantigen ELISA OD values had dual peaks, one in June and the other in October, whereas taeniid eggs were detected only from April to July. Fox pup faeces (n=51) collected around the dens used by the positive family were all coproantigen and taeniid egg negative except for 1 faeces. These results suggest that the prevalence and infection pressure in the study area were low and the infected family terminated the infection in the summer and acquired a new one in the early autumn, 1994. This monitoring method for fox infection will be a practical management tool for E. multilocularis infection, especially at the tourist spots in Hokkaido where close contact of fox with humans is frequently observed.