There is some evidence that pathology induced by heavy malaria infections (many oocysts) increases mortality of infected mosquitoes. However, there is little or no published evidence that documented changes in feeding behaviour associated with malaria infection also contribute to higher mortality of infected mosquitoes relative to uninfected individuals. We show here for the first time that, in a natural situation, infection by the sporozoites of the malaria parasite Plasmodium falciparum significantly reduced survival of blood-feeding Anopheles gambiae, the major vector of malaria in sub-Saharan Africa. To estimate feeding-associated mortality of infected mosquitoes, we compared the percentage of sporozoite infection in host-seeking mosquitoes caught before and after feeding. The infection rate was 12% for mosquitoes caught during the night as they were entering a tent to feed; however, only 7·5% of the surviving members of the same cohort caught after they had had the opportunity to feed were infected. Thus, Plasmodium falciparum sporozoites increased the probability of dying during the night-time feeding period by 37·5%. The increase in mortality was probably due to decreased efficiency in obtaining blood and by increased feeding activity of the sporozoite-infected mosquitoes that elicited a greater degree of defensive behaviour of hosts under attack.

To date, a high degree of polymorphism has been demonstrated at both the MSP1 and MSP2 loci in parasites from areas of stable malaria transmission. As a consequence, in such areas it is rare to find parasites of the same 2-locus genotype in more than 1 subject. We have studied MSP1 and MSP2 diversity in parasites collected from subjects with both symptomatic (n = 86) and asymptomatic (34) malaria living on the island of Santo, Vanuatu, an area of stable malaria transmission. Polymorphism at the MSP1 and MSP2 loci was considerably less than previously reported: only 5 MSP1 and 5 MSP2 alleles were detected and these showed no size variation within alleles. Santo is unique amongst the areas studied so far in that it is a small island at the limit of the malaria belt in the South Pacific. Thus, the evolution of the parasite population may have been affected by the small size and isolation of this island population. Moreover, limited parasite diversity may explain the unusually mild nature of Plasmodium falciparum disease on Santo. Islands have fascinated biologists for centuries and fuelled the advancement of evolutionary theory, since they are natural laboratories for the study of evolution. The simplicity of the Vanuatu P. falciparum population may facilitate the use and interpretation of sequence level analyses to address the mechanisms by which genetic diversity is generated and maintained in natural populations.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S–5S–18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.The nucleotide sequence data reported in this paper are available in the EMBL database with the accession number AJ238400.The commercial use of the diagnostic test presented here is protected by a patent. This patent is pending upon the application number P9801511.

The first long-term (27 month) survey of single species of Eimeria occurring in a population of Apodemus sylvaticus in the UK showed that Eimeria apionodes, E. hungaryensis and E. uptoni occurred throughout the period whilst E. sp. E. was only found during 4 months. In first-captured animals, overall prevalence of Eimeria spp. was 73%: a figure higher than previously published, but possibly an underestimate. Multiple infections (2 or 3 species) occurred in 34% of first-capture infected animals, but without significant associations. There were no significant differences of prevalence rates between the sexes, nor between adults and juveniles, except for E. uptoni which was more common in juveniles than in adults. E. hungaryensis and E. uptoni probably occurred as near-continuous infections whilst E. apionodes occurred more intermittently but with no long-term total immunity. Monthly prevalence data indicated ongoing trends over the 27 months. Prevalences of E. apionodes indicated an annual cycle, lowest in January–May and with a peak in October–November, correlating positively with both the number of animals and the percentage of juveniles in the host population and also with the environmental relative humidity. There was no consistent pattern for E. uptoni and an annual cycle for E. hungaryensis was not apparent.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0·5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

A statistically derived disease reaction index based on parasitological, clinical and haematological measurements observed in 309 5 to 8-month-old Boran cattle following laboratory challenge with Theileria parva is described. Principal component analysis was applied to 13 measures including first appearance of schizonts, first appearance of piroplasms and first occurrence of pyrexia, together with the duration and severity of these symptoms, and white blood cell count. The first principal component, which was based on approximately equal contributions of the 13 variables, provided the definition for the disease reaction index, defined on a scale of 0–10. As well as providing a more objective measure of the severity of the reaction, the continuous nature of the index score enables more powerful statistical analysis of the data compared with that which has been previously possible through clinically derived categories of non-, mild, moderate and severe reactions.

A cDNA library derived from mRNA of tachyzoites of Neospora caninum (NC-Liverpool strain) was screened with antisera from a cow naturally infected with N. caninum. The DNA sequence of 1 recombinant isolated predicted a significant protein sequence homology of the gene product to the 28 kDa (GRA2) antigen of Toxoplasma gondii. Studies on the N. caninum gene coding for this antigen demonstrated the presence of a single intron flanked by 2 exons; the gene was also highly expressed in culture-derived tachyzoites. The antigen was expressed in Escherichia coli; when injected into mice it stimulated the production of antibodies which detected a 29 kDa antigen of N. caninum. Secondary structure predictions made for the N. caninum protein showed support for several amphipathic helices separated by loops and turns. The available evidence indicates maintenance of protein secondary structure, and not DNA or amino acid sequence, has occurred during the evolution of GRA2 proteins in N. caninum and T. gondii.

Nitric oxide (NO) produced by cytokine-activated macrophages is reported to be cytotoxic against the helminth Schistosoma mansoni, although this is a controversial issue. Previous work in our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to soluble egg antigen (SEA) or to SWAP. Here we report that, in comparison to other S. mansoni antigenic preparations (SEA and SWAP), supernatants of PBMC cultivated with PIII possess higher concentrations of interleukin-10 (IL-10) and macrophage inflammatory protein (MIP-1α), concomitantly with lower concentrations of monocyte chemoattractant protein (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES). In the particular case of NO inhibition, supernatants of PBMC cultivated with PIII present decreased IL-10 levels. Altogether, these results indicate that IL-10, MIP-1α, MCP-1 and RANTES are distinctively important elements in the PIII modulating role, while NO seems to be pivotal in the regulation of granulomatous responses.

Changes in gene expression in Biomphalaria glabrata following infection with Schistosoma mansoni have been investigated using a modified differential display approach. RNA was extracted from ovotestis, mantle tissue and anterior nephridium of control and exposed snails at 2 time-points (4 h and 24 h) post-exposure and analysed by RNA fingerprinting. A number of transcripts were identified; some novel and some homologous to mRNAs in GenEMBL that were previously unknown in B. glabrata. Down regulation of one 241 bp mRNA expressed sequence fragment – with an open reading frame showing 48% identity to a cytochrome p450 over 80 residues – has been confirmed using semi-quantitative RT-PCR. Preliminary classification of B. glabrata cyp450 sequence shows it to fall into CLAN 2 of the cytochrome p450 superfamily. Differential display has been successful in identifying changes in gene expression in Biomphalaria glabrata upon infection with Schistosoma mansoni and promises to be a useful technique for the investigation of the interaction between host and parasite.

Argulus foliaceus, an obligate fish ectoparasite, can search for its hosts in both light and dark conditions and uses vision in the light. We have examined what searching mode is used at night, when the infection rate was at its highest, and which stimuli produced by the fish are most important. A change of illumination produced a clear difference in the searching behaviour of adult Argulus females. The mean swimming speed and the area explored were 3–4 times higher in the dark, when the parasite employed a cruising search strategy. This changed to an ambush (hover-and-wait) strategy in the light. The swimming activity is accompanied by changes in metabolic costs; the activity of the electron transport system being approximately 25% lower in the light. The most pronounced light-induced differences in host-searching behaviour took place in moderately hungry parasites (starved for 24–96 h). Less motivated (just having left a fish) or exhausted animals did not exhibit any clear differences in swimming speed. Among the external signals tested, fish smell, from both perch (Perca fluviatilis) and roach (Rutilus rutilus), induced an elevated swimming speed of the parasite. Periodic water movements caused similar but weaker effects. The effects of these stimuli were observed under both light and dark conditions. We conclude that host-searching behaviour of A. foliaceus is under internal (state of hunger) and external (illumination and host-induced signals) control and involves all its sensory equipment (vision, olfaction and mechanoreception). Perch (but not roach) reduced their swimming speed in the dark, which make them more susceptible to cruising Argulus. Thus the behavioural interplay between hosts and parasites can also influence the infection rate of A. foliaceus found on perch and roach in Finnish lakes.

Caffeine, which stimulates intracellular Ca2+ release channels known as ryanodine receptor (RyR) channels, induces contraction of individual muscle fibres dissociated from the trematode Schistosoma mansoni, and the turbellarians Dugesia tigrina and Procerodes littoralis. Caffeine is much more potent on S. mansoni fibres (EC50 0·7 mM) than those from D. tigrina or P. littoralis (3·2 mM and 4·6 mM, respectively). These caffeine-induced contractions are blocked by ryanodine, confirming the presence of functional RyR channels in these flatworm muscles. However, the contractions are not blocked by typical RyR channel blockers ruthenium red or neomycin, indicating that there may be important pharmacological differences between the RyR channels in this early-diverging phylum and those of later animals. These studies demonstrate that RyR channels are present in the muscle of these flatworms, and that the sarcoplasmic reticulum stores sufficient Ca2+ to support contraction.

In order to assess the infection risk of alveolar echinococcosis among urban residents of Sapporo, the capital of Hokkaido, Japan, a survey was conducted on fox distribution in the urban area and on the prevalence of Echinococcus multilocularis among the foxes. The fox distribution, evaluated from fox footprints left on the snow in parks and woodlands, and from locations of fox carcasses recorded by the Sapporo municipality, was concentrated along the border of the urban area and in the southwestern part of the city, facing the mountain. Fox faeces were collected around active fox dens, and analysed by a coproantigen detection assay and parasite egg examination for the Echinococcus infection. Thirty-three out of 155 faeces were coproantigen positive. Coproantigen-positive faeces were collected from 11 den sites (57·9% of total den sites), and all except 1 were located in the urban fringe. A high intensity of taeniid eggs (>100 eggs per 0·5 g) containing faeces were also collected in the 3 sites of them. Although Echinococcus infection in rodents was not observed from the necropsy of 23 rodents captured around active fox dens, arvicolid rodents, a suitable intermediate host for E. multilocularis, were captured in the urban fringe. Therefore, the urban fringe offers suitable conditions in which the life-cycle of E. multilocularis could be maintained. Prompt measures to control echinococcus infection should be taken, even in urban areas.

Factors constraining the evolution of host-specificity were investigated using a gastrointestinal parasitic nematode, Strongyloides ratti. S. ratti is a natural parasite of rats which can also reproduce, with decreased success, in laboratory mice. Observed host-specificity arose from lower establishment, reduced per capita fecundity and more rapid expulsion of parasites from mice relative to rats. Variation in the efficacy of thymus-dependent immunity between host species (rats and mice) was insufficient to explain the majority of the observed differences in parasite establishment and reproductive success. The role of natural selection in determining host-specificity was addressed using experimental selection followed by reciprocal fitness assays in both host species. Experimental selection failed to modify the host-specificity of S. ratti to any measurable degree, suggesting either a lack of genetic variation for this trait or the involvement of as yet unidentified factors underlying the differences in S. ratti fitness in rats and mice respectively. These results are discussed in relation to competing theoretical models of ecological specialization, host immunology and previous attempts to experimentally alter the host-specificity of parasitic nematodes