Cane toads (Bufo marinus) were introduced to Australia in 1935 and have since spread widely over the continent, generating concern regarding ecological impacts on native predators. Most Australian cane toad populations are infected with lung nematodes Rhabdias pseudosphaerocephala, a parasite endemic to New World (native-range) cane toad populations; presumably introduced to Australia with its toad host. Considering the high intensities and prevalence reached by this parasite in Australian toad populations, and public ardour for developing a control plan for the invasive host species, the lack of experimental studies on this host-parasite system is surprising. To investigate the extent to which this lungworm influences cane toad viability, we experimentally infected metamorph toads (the smallest and presumably most vulnerable terrestrial phase of the anuran life cycle) with the helminth. Infected toads exhibited reduced survival and growth rates, impaired locomotor performance (both speed and endurance), and reduced prey intake. In summary, R. pseudosphaerocephala can substantially reduce the viability of metamorph cane toads.

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

SSU ribosomal sequences of trypanosomes from Brazilian cattle and water buffalo were used to infer phylogenetic relationships between non-pathogenic T. theileri and allied species parasitic in artiodactyls. T. theileri trypanosomes from distinct geographical regions in Brazil and from other countries were tightly clustered into the ‘clade T. theileri’ distant from the ‘T. brucei clade’ of pathogenic parasites of artiodactyls, and also distinct from trypanosomes of other mammals. The existence of this monophyletic assemblage (T. theileri clade) composed only by isolates from artiodactyl species justifies the continued recognition of the subgenus T. (Megatrypanum) with T. theileri as its type species. Phylogenies based on SSU and ITS1 ribosomal sequences produced the same branching pattern with isolates from different mammalian hosts clustered in 5 lineages: A, related to water buffalo; B, C and D, to cattle; E, to fallow deer. The pattern of host specificity allied to some congruence between host and parasite phylogenies suggested association of these trypanosomes with their respective hosts. Segregation of cattle isolates into three lineages revealed an overall geographical structure. Moreover, positioning of trypanosomes infecting tabanids in the T. theileri clade is consistent with the role of these flies as important vectors of these trypanosomes.

Quantitative histological methods were applied in a study of the migratory route of schistosomula within the definitive mammal host. The observations are consistent with an entirely intravascular mode of migration in the direction of blood flow. They do not support a trans-diaphragm route. Schistosomula can be identified in low numbers in systemic organs, in the left side of the heart and in the venous compartment of the pulmonary circulation. They were not observed penetrating the diaphragm or the capsule of the liver. No histopathological evidence was found to suggest that the tissue responses of previously unexposed hosts affect migrating schistosomula at any stage during migration. These non-specific tissue responses were marked only in the skin phase of migration.

The author has summarized the history of discovery, the mechanism and the clinical significance of antibody-dependent enhancement (ADE) of HIV infection. ADE has two major forms: (a) complement-mediated antibody-dependent enhancement (C-ADE) and (b) complement-independent Fc receptor-dependent ADE (FcR-ADE). The most important epitope responsible for the development of C-ADE-mediating antibodies is present in the immunodominant region of gp41 while antibodies mediating FcR-ADE react mainly with V3 loop of gp120. There are at least three fundamentally different hypotheses for the explanation of ADE in vitro: (a) increased adhesion of HIV-antibody-(complement) complexes to FcR or complement receptor carrying cells; (b) facilitation of HIV-target cell fusion by complement fragment deposited on the HIV-virions and (c) complement activation products may have a non-specific stimulatory effect on target cells resulting in enhanced virus production. FcR-ADE and C-ADE have been measured in vitro mostly by using FcR-carrying and complement receptor-carrying cell lines, respectively; no efforts have been made to standardize these methods. Several data support the possible clinical significance of FcR-ADE and C-ADE: (a) Cross-sectional and longitudinal studies indicate a correlation between the amounts of FcR-ADE and C-ADE-mediating antibodies and clinical, immunological and virological progression of the HIV-disease; (b) ADE may facilitate maternal–infant HIV-1 transmission; (c) According to experiments in animal models, ADE are present and may modify the course of SIV (simian immunodeficiency) infection as well. The author raises a new hypothesis on the mechanism of the in vivo effect of C-ADE. According to the hypothesis, C-ADE-mediating antibodies exert their effect through enhancement of HIV propagation and consequent facilitation of the progression of HIV disease. Finally, according to observations from animal experiments and human clinical trials it cannot be excluded that ADE-mediating antibodies may develop, diminish the beneficial effect or may be harmful in volunteers vaccinated with HIV-1 candidate vaccines.

The Cryptosporidium ITS1, 5·8S and ITS2 rDNA regions from a number of Cryptosporidium isolates from different hosts and geographical areas were cloned and sequenced in order to investigate the extent of sequence heterogeneity between human and cattle-derived isolates from different geographical locations and also between isolates of Cryptosporidium from different hosts such as cats, pigs, mice and a koala. Calf-derived isolates from different continents were virtually identical as were human-derived isolates from the UK and Australia. Genetic differences between Cryptosporidium isolates were extensive and were in fact greater than the level of nucleotide divergence between Toxoplasma gondii and Neospora caninum rDNA sequences. Based on the sequence information derived from this study, PCR–RFLP of the ITS1 region was undertaken in order to directly amplify and genotype Cryptosporidium isolates from different hosts. This PCR–RFLP approach can now be used for molecular epidemiology studies, circumventing the need for costly sequencing and allowing a wider range of genetically different isolates to be examined.

Cystic echinococcosis, caused by Echinococcus granulosus, is an emerging disease in many parts of the world and, in particular, in eastern Europe and the former Soviet Union. This paper examines the abundance of infection of E. granulosus in the definitive host in southern Kazakhstan. Observed data are fitted to a mathematical model in order to decide if the parasite population is partly regulated by definitive host immunity and to define parameters in the model. Such data would be useful to develop simulation models for the control of this disease. Maximum likelihood techniques were used to define the parameters and their confidence limits in the model and the negative binomial distribution was used to define the error variance in the observed data. The results indicated that there were 2 distinct populations of dogs in rural Kazakhstan which had significantly different exposures to E. granulosus. Farm dogs, which are closely associated with livestock husbandry, particularly sheep rearing, had a relatively high mean abundance of 631 parasites per dog and a prevalence rate of approximately 23%. The best fit to the model indicated that there was significant herd immunity in the dog at this infection pressure. In contrast, village dogs which were more likely to be kept as pets had a much lower mean abundance of parasites of only 27 parasites per dog and a lower prevalence of 5·8%. With this village population of dogs, the best fit indicated negligible herd immunity.

The sexual development of the microgametocyte of Plasmodium yoelii nigeriensis may be subdivided into microgametogenesis which includes exflagellation, dispersal of the gametes and fertilization. Under our experimental conditions microgametogenesis takes about 8–15 min at 20 °C, the duration of this period being inversely related to temperature. Exflagellation takes less than 1 min, subsequent dispersal of gametes may continue for 40 min. We find that exflagellation is totally inhibited in vitro by temperatures of 30 °C and above, and by certain invertebrate tissue culture media. Exflagellation may occur within a persistent host cell plasmalemma, which seriously impedes the escape of the 8 microgametes. Microgametes move by sinusoidal or helical waves which may be rapid (10 waves/s), slow (< 1 wave/s) or they may be immobile. Microgamete activity, which is alternately rapid/slow or slow/immobile, declines linearly with time. Fertilization which takes less than 1 min results from characteristic behavioural changes by the microgamete; vibratory waves are responsible for the penetration of the macrogamete by the microgamete. The microgamete completely enters the macrogamete and therein continues its cyclical activity.

The encapsulated embryos of platyhelminths may be retained and complete their development in utero in a range of circumstances. However, hatching within the parent (the criterion of ovoviviparity) is relatively rare and larvae generally emerge only after deposition. Viviparity is characterized by the nutritional dependency of the unencapsulated larva upon the parent, but in several cases larvae retained within a shell also receive parental nutrients during intra-uterine development. Uptake of exogenous nutrients via shell pores occurs in Schistosoma mansoni but the eggs, which gain all the advantages of intra-uterine retention, are supported by host nutrients.

Intra-uterine larval development avoids the hazards of development in the external environment and eliminates the time delay between oviposition and infection. Deposition of immediately infective offspring may be concentrated in time and space to exploit periods of host vulnerability. The control and precision of transmission is illustrated by examples in which the opportunity for invasion is restricted because of either host behaviour or environmental instability. This strategy has been an important factor in the evolution of polystomatid monogeneans, and its effectiveness is demonstrated by comparison of the life-cycles of Polystoma integerrimum and Pseudodiplorchis americanus. Ovoviviparity also increases reproductive potential in some polystomatids by extending the period of multiplication and by increasing established populations through internal re-infection. In Eupolystoma alluaudi, the capacity for ovoviviparity is programmed into larval development and this regulates population growth within individual hosts.

Migration by nematode larvae through the tissues of their mammalian hosts can cause considerable pathology, and yet the evolutionary factors responsible for this migratory behaviour are poorly understood. The behaviour is particularly paradoxical in genera such as Ascaris and Strongylus in which larvae undergo extensive migrations which begin and end in the same location. The orthodox explanation for this apparently pointless behaviour is that a tissue phase is a developmental requirement following the evolutionary loss of skin penetration or intermediate hosts. Yet tissue migration is not always necessary for development, and navigation and survival in an array of different habitats must require costly biochemical and morphological adaptations. Migrating larvae also risk becoming lost or killed by the host. Natural selection should therefore remove such behaviour unless there are compensating benefits. Here we propose that migration is a selectively advantageous life-history strategy. We show that taxa exploiting tissue habitats during development are, on average, bigger than their closest relatives that develop wholly in the gastrointestinal tract. Time to reproduction is the same, indicating that worms with a tissue phase during development grow faster. This previously unsuspected association between juvenile habitat and size is independent of any effects of adult habitat, life-cycle, or host size, generation time or diet. Because fecundity is intimately linked with size in nematodes, this provides an explanation for the maintenance of tissue migration by natural selection, analogous to the pre-spawning migrations of salmon.

1. Experimental infections in mice showed that the larvae of Toxocara canis, T. mystax, Ascaris devosi, A. columnaris and Toxascaris transfuga reached the brain of mice; the larvae of Ascaris lumbricoides, A. suum, Parascaris equorum and Toxascaris leonina were not recovered from the brain. The larvae of T. canis, T. mystax, T. transfuga and A. columnaris remained hi the brain of mice for several months.

2. Larvae reaching the brain produced characteristic haemorrhages on the surface of the cerebral hemispheres in the early stages of infection. It was concluded that the larvae reach the brain via the arterial blood stream, leave the arteries at the point where their diameter approximates that of the larvae, i.e. mostly on the surface of the brain, and penetrate into the brain from the subarachnoid space and chorioidal tissues.

3. The larvae of T. canis were found to occur in the brain of mice in relatively greater numbers than the larvae of other species, but only very rarely caused nervous symptoms. The larvae of T. canis and T. mystax showed no growth in the brain.

4. The larvae of A. columnaris (skunk) frequently caused nervous symptoms in mice, the effect appeared to result from traumatic damage due to the relatively large size attained by these larvae about 3 weeks after infection.

5. The brain of infected mice showed very slight changes consequent upon infection with larvae of T. canis. These larvae moved actively through the tissues; they incited little or no cellular reaction, but left haemorrhagic tracks. The larvae of A. columnaris also moved actively; when in the extended state they were usually found in normal tissue; when coiled, they were often associated with a necrotic focus infiltrated with leucocytes.

6. After experimental infections of dogs with larvae of T. canis, two out of twelve infected animals harboured larvae in the brain. No natural infections with these larvae were found in the brains of dogs and cats. After experimental infection, larvae of T. canis were found in the brain of mice, rats and guinea-pigs, but not of rabbits.

7. Larvae of A. suum were recovered from the cerebrum of one pig suffering from posterior paralysis, but not in an experimentally infected pig.

8. No larvae of P. equorum were found in the brain of foals in natural and experimental infections.

Encapsulation of the benzimidazole albendazole in multilamellar liposomes results in a preparation in which this normally insoluble anti-hydatid drug is well solublilized in aqueous media. The high entrapment efficiency observed (75–87%) and the stability of the formulation make this a promising delivery vehicle for improved chemotherapy with albendazole. In particular, the high degree of association with phospholipid may give rise to increased oral bioavailability. Oral adminisration of albendazole in liposomes led to increased concentration and/or altered metabolism of albendazole sulphoxide (ABZSX) in liver and/or plasma in non-infected Wistar rats. Results from experiments using cotton rats (Sigmodon hispidus) infected with metacestodes of Echinococcus multilocularis show that entrapment within liposomes clearly increases the uptake of albendazole via the oral route. This was reflected by increased levels of albendazole and the two major metabolites in plasma, liver and cyst homogenate when a dose of liposomal albendazole (35 mg/kg) was given orally compared to free albendazole at 50 mg/kg. There was a 75–94% reduction in biomass of the metacestode and a significant increase in survival time for the animals treated with liposome entrapped albendazole. A clear difference in distribution of albendazole and its metabolites in the liver and the metacestode tissues in the presence of cimetidine indicated that the latter has a profound effect on the metabolism of albendazole. There appeared to be a synergistic interaction between albendazole and cimetidine, since the metabolism of albendazole was markedly altered in the combined cimetidine/liposome–albendazole group, and higher therapeutic effect was observed. These findings indicate potential both for improvement of treatment of larval E. multilocularis infection and for reduction of albendazole dose levels.

Every few years a large proportion of the feral sheep on Hirta, St Kilda die due to food shortage. The effects of malnutrition are exacerbated by gastrointestinal nematodes. As found in sheep flocks in mainland Britain, Teladorsagia circumcincta has long been considered the predominant and most pathogenic nematode species in all age classes of Soay sheep. Previous research indicated that intensity of this species showed a negative association with host age and comprised 75% of the entire gastrointestinal burden. Here we present new data that show Trichostrongylus axei and Trichostrongylus vitrinus to be the predominant worm pathogens in young Soay sheep. In the present study, Trichostrongylus spp. burdens declined with host age whereas T. circumcincta actually increased in burden over the first few age classes. Also, male hosts had significantly higher burdens of Trichostrongylus spp. than females, with this genus making up a higher proportion of the strongyle egg producing community in male hosts than female hosts. These new findings raise questions concerning our previous interpretation of the main nematode species contributing to strongyle egg count in the population, and the contrasting infection patterns of these nematode species in unmanaged St Kilda Soay sheep compared with domestic sheep in mainland Britain.

Given that numerous amphibians are suffering population declines, it is becoming increasingly important to examine the relationship between disease and environmental disturbance. Indeed, while many studies relate anthropogenic activity to changes in the parasitism of snails and fishes, little is known of the impact on the parasites of amphibians, particularly from agriculture. For 2 years, the parasite communities of metamorphic northern leopard frogs from 7 agricultural wetlands were compared with those from 2 reference wetlands to study differences in parasite community diversity and abundance of various species under pristine conditions and 3 categories of disturbance: only agricultural landscape, only pesticides, and agricultural landscape with pesticides. Agricultural (and urban) area was negatively related to species richness, and associated with the near absence of adult parasites and species that infect birds or mammals. We suggest that agriculture and urbanization may hinder parasite transmission to frogs by limiting access of other vertebrate hosts of their parasites to wetlands. The only parasite found at all localities was an unidentified echinostome infecting the kidneys. This parasite dominated communities in localities surrounded by the most agricultural land, suggesting generalist parasites may persist in disrupted habitats. Community composition was associated with dissolved organic carbon and conductivity, but few links were found with pesticides. Pollution effects may be masked by a strong impact of land use on parasite transmission.

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1α, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1α. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Vaccination against complex metazoan parasites has become a reality with the development and registration of recombinant protein-based vaccines against the cattle tick Boophilus microplus and the sheep cestode Taenia ovis. Progress towards the development of similar vaccines against gastrointestinal nematodes, primarily of ruminants, is outlined within a framework of defining the practical requirements for successful vaccination, antigen selection, recombinant protein production and antigen delivery, be it mucosal delivery or DNA vaccination. Antigen selection strategies include the fractionation of complex, but protective, parasite extracts, the use of antibody probes, evaluation of excretory-secretory components and gut-expressed hidden antigens as well as antigens targeted on the basis of function such as enzyme activity. The difficulties being encountered in recombinant protein production and their solution are discussed as are the requirements for successful antigen delivery. Recent technological developments such as the use of functional genomics to identify new vaccine candidates and DNA vaccination to present the selected antigen to the host immune system are discussed and are anticipated to have a profound effect on vaccine development in the future.

During an investigation, extending over several years, of the blood of numerous animals found locally, I have met with numerous parasites most of which are well known, but some, at least to me, are new.

Lambs aged 48–150 days which had been injected with extracts enriched in a functional antigen (contortin) obtained from adult Haemonchus contortus developed specific circulating antibodies and were less susceptible to haemonchosis when challenged 1 month later with a single dose of 20000–25000 infective 3rd-stage larvae. Sera from 18 out of a total of 19 lambs injected with the extract contained precipitating antibodies to 2–5 components of the extract. None of these lambs died. The one extract-injected lamb which did not develop antibodies and 9 of the 13 lambs used as controls died of haemonchosis. The average weight of worms recovered was 1·45 g from the immune lambs and 5·72 g from the non-immune lambs.

The mechanism by which small animals such as rodents resist or eliminate nematode parasites requires mucosal in flammation as the final effector of the immune response. The resulting freedom from chronic infection may be worth the price of short-term illness. Putative vaccines which attempt to enhance the natural effect will have to take into account the inflammatory cost to the host. Human helminthiases involve a more stable equilibrium between host and parasite. The medical literature on hookworm disease and clinical ascariasis describes, for the former, some chronic inflammatory effects correlated with worm burden, but for the latter a less quantified or predictable set of detrimental effects. We describe a current, systematic study of the inflammatory response to whipworm infection, in which anaemia, growth retardation and intestinal leakiness are viewed as predictable consequences related to infection intensity. There is evidence for the absence of cell-mediated immunopathology. However, a specific, IgE-mediated local anaphylaxis may, at least partly, mediate the deleterious effects. Increased numbers of mucosal macrophages may also contribute to the chronic, systemic effects through their output of cytokines. Similar attempts to show the mechanisms of pathogenesis and quantify the effects of hookworm disease should be undertaken.