The salivary glands are the organs of osmoregulation in ticks and, as such, are critical to the biological success of ticks both during the extended period off the host and also during the feeding period on the host. Absorption of water vapour from unsaturated air into hygroscopic fluid produced by the salivary glands permit the tick to remain hydrated and viable during the many months between blood-meals. When feeding, the tick is able to return about 70% of the fluid and ion content of the blood-meal into the host by salivation into the feeding site. This saliva also contains many bioactive protein and lipid components that aid acquisition of the blood-meal. The salivary glands are the site of pathogen development and the saliva the route of transmission. The importance of the multifunctional salivary glands to tick survival and vector competency makes the glands a potential target for intervention. Here we review the cell biology of tick salivary glands and discuss the application of new approaches such as expressed sequence tag projects and RNA interference to this important area in the field of tick and tick-borne pathogen research.

Plasmodium falciparum-infected erythrocytes (IRBC) synthesize 3 histidine-rich proteins: HRP-I or the knob-associated HRP, HRP-II and HRP-III or SHARP. In order to distinguish these proteins immunochemically we prepared monoclonal antibodies which react with HRP-I, HRP-II and HRP-III, and rabbit antisera against synthetic peptides derived from the HRP-II and HRP-III sequences. A comparative analysis of diverse P. falciparum parasites was made using these antibodies and immunoprecipitation or Western blotting. HRP-I (Mr 80000–115000) was identified in all knob-positive P. falciparum parasites including isolates examined directly from Gambian patients. However, this protein was of lower abundance in these isolates and in 6 knob-positive, culture-adapted parasites compared to Aotus monkey-adapted parasites or culture-adapted parasites studied previously. HRP-II (Mr 60000–105000) was identified in all P. falciparum parasites regardless of knob-phenotype, and was recovered from culture supernatants as a secreted water-soluble protein. Within IRBC, HRP-II was found as a complex of several closely spaced bands. Cell surface radio-iodination of IRBC from several isolates and immunoprecipitation with a rabbit antiserum against the HRP-II repeat sequence identified HRP-II as a surface-exposed protein. Like HRP-I, the abundance of HRP-II was lower in the Gambian isolates than with Aotus monkey-adapted parasites studied earlier. Neither HRP-I nor HRP-II were identified in a knob-positive isolate of P. malariae collected from a Gambian patient. Analogues of these HRP were also absent from asexual parasites of diverse primate and murine malaria species screened with this panel of antibodies. HRP-III (Mr 40000–55000) was distinguished by its lower apparent size and by specific reaction with rabbit antibody against its 5-mer repeat sequence. HRP-III was of lowest abundance compared with the other two HRP. These antibody reagents and distinguishing properties should prove useful in studies on the separate functions of the 3 P. falciparum HRP.

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting many intensively-reared livestock, especially poultry. The chicken is host to 7 species of Eimeria that develop within intestinal epithelial cells and produce varying degrees of morbidity and mortality. Control of coccidiosis by the poultry industry is dominated by prophylactic chemotherapy but drug resistance is a serious problem. Strongly protective but species-specific immunity can be induced in chickens by infection with any of the Eimeria spp. At the Institute of Animal Health in Houghton, UK in the 1980s we showed that all 7 Eimeria spp. could be stably attenuated by serial passage in chickens of the earliest oocysts produced (i.e. the first parasites to complete their endogenous development) and this process resulted in the depletion of asexual development. Despite being highly attenuated, the precocious lines retained their immunizing capacity. Subsequent work led to the commercial introduction of the first live attenuated vaccine, Paracox®, that has now been in use for 20 years. As much work still remains to be done before the development of recombinant vaccines becomes a reality, it is likely that reliance upon live, attenuated vaccines will increase in years to come.

Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four in-dependent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.

The taxonomic status of Echinococcus, an important zoonotic cestode genus, has remained controversial, despite numerous attempts to revise it. Although mitochondrial DNA (mtDNA) has been the source of markers of choice for reconstructing the phylogeny of the genus, results derived from mtDNA have led to significant inconsistencies with earlier species classifications based on phenotypic analysis. Here, we used nuclear DNA markers to test the phylogenic relationships of members of the genus Echinococcus. The analysis of sequence data for 5 nuclear genes revealed a significantly different phylogeny for Echinococcus from that proposed on the basis of mitochondrial DNA sequence data, but was in agreement with earlier species classifications. The most notable results from the nuclear phylogeny were (1) E. multilocularis was placed as basal taxon, (2) all genotypes of Echinococcus granulosus grouped as a monophyletic entity, and (3) genotypes G8 and G10 clustered together. We conclude that the analysis of nuclear DNA data provides a more reliable means of inferring phylogenetic relationships within Echinococcus than mtDNA and suggest that mtDNA should not be used as the sole source of markers in future studies where the goal is to reconstruct a phylogeny that does not only reflect a maternal lineage, but aims to describe the evolutionary history at species level or higher.

We present observational and experimental evidence that cycles of the Lyme disease spirochaete, Borrelia burgdorferi s.l., can be maintained by sheep in the virtual absence of alternative hosts. A 2-year field study in upland moorland habitats of northwest UK established that sheep feed up to 80% of larval, >99% of nymphal and all of the adult female tick (Ixodes ricinus) population. Infection prevalence of B. burgdorferi in questing ticks reaches over 20%, but amplification of infection occurs principally as nymphs (20- to 30-fold), rather than larvae (4- to 7-fold), feed on sheep, and transmission from sheep to ticks occurred only during peak tick abundance in May and September. Experimental transmission studies confirmed that sheep, previously exposed to infected ticks on the moorland site, do not support systemic infections of B. burgdorferi, but they can transmit localized infections from infected to uninfected ticks co-feeding at the same site on the sheep's body.

Congenital infection with Toxoplasma gondii is an important cause of abortion in sheep worldwide. The cat is the definitive host of the parasite, and infected cats may shed millions of oocysts in their faeces resulting in extensive environmental contamination and an important source of infection for grazing herbivorous animals. Studies looking at development of specific antibodies in sheep, as an indicator of exposure to T. gondii, have shown that there is an increase in seroprevalence associated with age indicating that most infections in sheep occur following birth. The stage of gestation when transplacental transmission of T. gondii to the developing foetus occurs is critical in determining the clinical outcome. The importance of endogenous transplacental transmission in persistently infected ewes and its clinical importance is a subject of current debate. Ewes infected prior to mating develop immune responses that help protect against disease in a subsequent pregnancy and also against experimental challenge administered during pregnancy. Both innate and adaptive immune responses are activated following T. gondii infection and experiments involving the chronic cannulation of peripheral lymph nodes in sheep have allowed the dynamics of the immune responses to be analysed in real time. A live vaccine, Toxovax® is the only commercially available vaccine worldwide to protect against congenital toxoplasmosis.

Mice which had been pre-treated with killed Corynebacterium parvum given intravenously or intraperitoneally, but not subcutaneously, were completely resistant ot infection withBabesia microti or B. rodhaini, and protected from death caused by Plasmodium vinckeior P. chabaudiinfection. There is evidence that the parasites died within circulating erythrocytes. This occurred much too soon for a specific antibody response to be evoked, and no antibody could be detected by the indirect fluorescent-anitbody technique. Thus it is suggested that a non-secific soluble mediator may play an important role in the protection observed.

Molecular approaches are being used increasingly for epidemiological studies of visceral and cutaneous leishmaniases. Several molecular markers resolving genetic differences between Leishmania parasites at species and strain levels have been developed to address key epidemiological and population genetic questions. The current gold standard, multilocus enzyme typing (MLEE), needs cultured parasites and lacks discriminatory power. PCR assays identifying species directly with clinical samples have proven useful in numerous field studies. Multilocus sequence typing (MLST) is potentially the most powerful phylogenetic approach and will, most probably, replace MLEE in the future. Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. Population genetic studies by MLMT revealed geographical and hierarchic population structure in L. tropica, L. major and the L. donovani complex. The existence of hybrids and gene flow between Leishmania populations suggests that sexual recombination is more frequent than previously thought. However, typing and analytical tools need to be further improved. Accessible databases should be created and sustained for integrating data obtained by different researchers. This would allow for global analyses and help to avoid biases in analyses due to small sample sizes.

Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.

Experiments were undertaken to investigate the factors which influence the transmission dynamics of Gyrodactylus bullatarudis within populations of laboratory guppies. The parasites possess a number of biological attributes which are almost unique amongst helminth parasites of vertebrates. These include the ability to reproduce viviparously and directly on the surface of the host, the ability to survive death of the host (for a short period of time) and the ability to transfer between hosts (despite the absence of a specialized transmission stage in the parasite's life-cycle). Long-term laboratory experiments demonstrated the inability of the parasite to persist within populations of the host in the absence of the continual introduction of naive susceptible fish. With regular addition of susceptible fish, the parasite population exhibited recurrent epidemic behaviour. The magnitudes of the epidemics and the time interval between them, were dependent on the rate at which fish were added to the populations. The parasite was over-dispersed in its distribution within the experimental fish populations and was a significant cause of host mortality (in a manner related to parasite burden). The experimental results suggest that acquired resistance to infection is an important factor determining epidemic behaviour.

Fasciola hepatica is a pathogenic trematode parasite of ruminants with a global distribution. Here, we briefly review the current epidemiology of bovine fasciolosis in Europe and discuss the progress made over the last decade in the diagnosis, impact on production and prediction of F. hepatica in cattle. Advances in diagnosis have led to significantly improved coprological and serological methods to detect presence of infection. Diagnostic test results have been correlated with intensity of infection and associated production losses, unravelling the impact on carcass weight and milk yield in modern cattle production systems. The economic impact of fasciolosis may, however, go beyond the direct impacts on production as evidence shows that F. hepatica can modulate the immune response to some co-infections. Control of bovine fasciolosis remains hampered by the limitations of the currently available flukicidal drugs: few drugs are available to treat dairy cows, many have low efficacies against juvenile stages of F. hepatica and there is evidence for the development of drug resistance. This makes research into the prediction of risk periods, and thus the optimum application of available drugs more pertinent. In this field, the recent research focus has been on understanding spatial risk and delivering region-specific spatial distribution maps. Further advances in epidemiological and economic research on bovine fasciolosis are expected to deliver farm-specific economic assessments of disease impact, to leverage non-chemotherapeutic management options and to enhance a more targeted use of anthelmintics.

Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4–6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA ‘endgame scenarios’, where test and treat strategies become more cost effective and deliverable.

The structure and invasive behaviour of extracellular erythrocytic merozoites prepared by a cell sieving method have been studied with the electron microscope.

Free merozoites contain organelles similar to those described in late schizonts of Plasmodium knowlesi. Their surface is lined by a coat of short filaments. On mixing with fresh red cells, merozoites at first adhere, then cause the red cell surface to invaginate rapidly, often with the formation of narrow membranous channels in the red cell interior. As the merozoite enters the invagination it forms an attachment by its cell coat to the rim of the pit, and finally leaves this coat behind as it is enclosed in a red cell vacuole. Dense, rounded intracellular bodies then move to the merozoite periphery, and apparently rupture to cause further localized invagination of the red cell vacuole. The merozoite finally loses its rhoptries, the pellicle is reduced to a single membrane and the parasite becomes a trophozoite. Invasion is complete by 1 min after adhesion, and the trophozoite is formed by 10 min.

Schistosoma mansoni is one of the most abundant infectious agents of humankind. Its widespread distribution is permitted by the broad geographic range of susceptible species of the freshwater snail genus Biomphalaria that serve as obligatory hosts for its larval stages. Molecular phylogenetic studies suggest that Schistosoma originated in Asia, and that a pulmonate-transmitted progenitor colonized Africa and gave rise to both terminal-spined and lateral-spined egg species groups, the latter containing S. mansoni. Schistosoma mansoni likely appeared only after the trans-Atlantic dispersal of Biomphalaria from the Neotropics to Africa, an event that, based on the present African fossil record, occurred only 2–5 million years ago. This parasite became abundant in tropical Africa and then entered the New World with the slave trade. It prospered in the Neotropics because a remarkably susceptible and productive host, B. glabrata, was widely distributed there. Indeed, a snail similar to B. glabrata may have given rise to the African species of Biomphalaria. Schistosoma mansoni has since spread into other Neotropical Biomphalaria species and mammalian hosts. The distribution of S. mansoni is in a state of flux. In Egypt, S. mansoni has nearly completely replaced S. haematobium in the Nile Delta, and has spread to other regions of the country. A susceptible host snail, B. straminea, has been introduced into Asia and there is evidence of S. mansoni transmission in Nepal. Dam and barrage construction has lead to an epidemic of S. mansoni in Senegal, and the parasite continues its spread in Brazil. Because of competition with introduced aquatic species and environmental changes, B. glabrata and consequently S. mansoni have become less abundant on the Caribbean islands. Control of S. mansoni using praziquantel and oxamniquine has reduced global prevalence but control is difficult to sustain, and S. mansoni can develop tolerance/resistance to praziquantel, raising concerns about its future efficacy. Because of legitimate environmental concerns, snail control is unlikely to be an option in future control efforts. Global warming will impact the distribution of Biomphalaria and S. mansoni, but the magnitude and nature of the effects are poorly understood.

RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

Recently, a novel human rickettsiosis, namely Atlantic rainforest spotted fever, was described in Brazil. We herein report results of a survey led around the index case in an Atlantic rainforest reserve in Peruibe municipality, southeastern Brazil. A Rickettsia parkeri-like agent (Rickettsia sp. Atlantic rainforest genotype) and Ricketsia bellii were isolated from adult Amblyomma ovale ticks collected from dogs. Molecular evidence of infection with strain Atlantic rainforest was obtained for 30 (12·9%) of 232 A. ovale adult ticks collected from dogs. As many as 88·6% of the 35 examined dogs had anti-Rickettsia antibodies, with endpoint titres at their highest to R. parkeri. High correlation among antibody titres in dogs, A. ovale infestations, and access to rainforest was observed. Amblyomma ovale subadults were found predominantly on a rodent species (Euryoryzomys russatus). From 17 E. russatus tested, 6 (35·3%) displayed anti-Rickettsia antibodies, with endpoint titres highest to R. parkeri. It is concluded that Atlantic rainforest genotype circulates in this Atlantic rainforest area at relatively high levels. Dogs get infected when bitten by A. ovale ticks in the forest, and carry infected ticks to households. The role of E. russatus as an amplifier host of Rickettsia to A. ovale ticks deserves investigation.

Cases of human Ascaris infection occur sporadically in areas such as N. America and Western Europe, where this parasite is thought to be non-endemic. Clinical case histories suggest that many of these cases may be cross-infections from pigs. I describe patterns of variation in the ribosomal DNA of Ascaris from 9 such cases. For comparative purposes, I also describe patterns of variation in parasites obtained from pigs and humans from worldwide locations. A Hae III restriction site distinguishes two classes of rDNA repeats; repeats bearing this restriction site were found in > 96% of parasites from pig populations worldwide and in all 9 worms from humans in N. America. In contrast, repeats bearing this restriction site were detected in < 2% of parasites from humans in endemic areas. The molecular data clearly incriminate pigs as the source of infection in the N. American cases. I discuss evolutionary and public health implications of incomplete host fidelity in Ascaris.

A review of the Giardia duodenalis sequences currently available on the GenBank database was completed to compare the different genotyping loci (small subunit ribosomal DNA, glutamate dehydrogenase, triose-phosphate isomerase and beta giardin) for their ability to discern assemblage and subassemblage groups and infer phylogenetic relationships. In total, 405 Giardia duodenalis sequences were sorted and aligned to examine the substitutions within and between the assemblages – A and B (zoonotic), C and D (dogs), E (livestock), F (cats) and G (rodents). It was found that all of the genes could reproducibly group isolates into their assemblages and that the AI/AII subassemblage groups were robust and identifiable at all loci. However, the assemblage B subgroups were not reproducible at half of the loci (small subunit ribosomal DNA and beta giardin), not due to their conserved nature, but because there was insufficient sequence data of reference isolates available for comparison. It is anticipated that further investigation of these loci may reveal the core subgroups of this medically important and zoonotic assemblage and also those of others. The closer, more recent, phylogenetic relationships amongst the assemblages appear to be resolved; however, more sequence data from the current loci, and possibly new loci, will be required to establish the remaining relationships.