A previous analysis of tick infestation patterns on rodents in Slovakia suggested that the key to the focal distribution of western-type tick-borne encephalitis virus (TBEv) in Europe is the geographically variable degree of synchrony in the seasonal activity of larval and nymphal Ixodes ricinus ticks. This prediction is here tested by examining records, from 7 different countries, of the seasonal variation in the abundance of larvae and nymphs feeding on rodents or questing on the vegetation. Larvae consistently started feeding and questing earlier in the year at sites within TBEv foci than elsewhere, so that they appeared in the spring as soon as nymphs were active. Such larval–nymphal synchrony is associated with a rapid fall in ground-level temperatures from August to October as revealed by the satellite-derived index of Land Surface Temperature (LST). Likewise, of 1992 pixels sampled on a grid across Europe, the 418 that fell within TBEv foci were characterized by a higher than average rate of autumnal cooling relative to the peak midsummer LST. It is proposed that such a seasonal temperature profile may cause unfed larvae to pass the winter in quiescence, from which they emerge synchronously with nymphs in the spring.

The Global Burden of Disease caused by the 3 major intestinal nematodes is an estimated 22·1million disability-adjusted life-years (DALYs) lost for hookworm, 10·5million for Ascaris lumbricoides, 6·4million for Trichuris trichiura, and 39·0million for the three infections combined (as compared with malaria at 35·7million) (World Bank, 1993; Chan et al. 1994); these figures illustrate why some scarce health care resources must be used for their control. Strongyloides stercoralis is the fourth most important intestinal worm infection; its nutritional implications are discussed, and the fact that its geographic distribution needs further study is emphasized. Mechanisms underlying the malnutrition induced by intestinal helminths are described. Anorexia, which can decrease intake of all nutrients in tropical populations on marginal diets, is likely to be the most important in terms of magnitude and the probable major mechanism by which intestinal nematodes inhibit growth and development. We present a revised and expanded conceptual framework for how parasites cause/aggravate malnutrition and retard development in endemic areas. Specific negative effects that a wide variety of parasites may have on gastrointestinal physiology are presented. The synergism between Trichuris and Campylobacter, intestinal inflammation and growth failure, and new studies showing that hookworm inhibits growth and promotes anaemia in preschool (as well as school-age) children are presented. We conclude by presenting rationales and evidence to justify ensuring the widest possible coverage for preschool-age children and girls and women of childbearing age in intestinal parasite control programmes, in order to prevent morbidity and mortality in general and specifically to help decrease the vicious intergenerational cycle of growth failure (of low-birth-weight/intrauterine growth retardation and stunting) that entraps infants, children and girls and women of reproductive age in developing areas.

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

An endemic transmission cycle of Babesia microti was discovered in Colorado in the foothills of the Rocky Mountains. B. microti were found by PCR in 4 of 25 Ixodes spinipalpis tick pools tested (a 3·2% minimum infection rate) and in 87% (13 of 15) of Microtus ochrogaster (the prairie vole) spleen and blood samples. Using naturally infected I. spinipalpis collected from wild-caught M. ochrogaster as vectors, B. microti and Borrelia bissettii were successfully transmitted to laboratory-born M. ochrogaster. Neither I. spinipalpis, nor M. ochrogaster (the prairie vole) have been previously reported as a vector or a reservoir host of B. microti. Unlike the east coast of the United States where Peromyscus leucopus is an important reservoir for B. microti, evidence for Peromyscus spp. (neither P. maniculatus nor P. difficilis) as B. microti reservoirs was not found in this study.

The vectorial competence of Phlebotomus sergenti for 3 Old World species of Leishmania, L. tropica, L. major and L. donovani, was investigated in vivo and by in vitro midgut binding assays using living promastigotes and purified lipophosphoglycan (LPG). P. sergenti consistently showed a high specificity for L. tropica strains, which were able to develop mature, potentially transmissible infections. The loss of infection with L. major and L. donovani correlated with the excretion of the digested bloodmeal. These strains were able to produce sustained infections in the midguts of their appropriate vectors, P. papatasi and P. argentipes, respectively. In in vitro binding assays, a significantly higher number of L. tropica procyclic promastigotes attached to the midgut lining of P. sergenti, compared to those of L. major and L. donovani (P < 0·05). The prediction that the species specificity of midgut attachment is controlled by polymorphic structures on the parasite LPG was supported by the finding that P. sergenti midguts were intensely stained following incubation with purified phosphoglycan (PG) from L. tropica compared with PGs from L. major or L. donovani. The results provide further evidence that LPG structural polymorphisms are driven by the species diversity of molecules present on the sandfly midgut that function as parasite attachment sites.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3α, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6·6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co- infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5–14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Endosymbiotic bacteria of the genus Wolbachia infect a number of invertebrate species in which they induce various alterations in host reproduction, mainly cytoplasmic incompatibility (CI). In contrast to most other maternally transmitted parasites, manipulation of host reproduction makes the spread of Wolbachia possible even if they induce a physiological cost on their hosts. Current studies have shown that fitness consequences of Wolbachia infection could range from positive (mutualist) to negative (parasitic) but, in most cases, Wolbachia do not have strong deleterious effects on host fitness and the status of association remains unclear. Here, we show that in the Drosophila parasitoid wasp Leptopilina heterotoma, Wolbachia infection has a negative impact on several host fitness traits of both sexes. Fecundity, adult survival and locomotor performance are significantly reduced, whereas circadian rhythm, development time and offspring sex-ratio are not affected. Although the cost of bacterial infection can be overcome by effects on host reproduction i.e. cytoplasmic incompatibility, it could influence the spread of the bacterium at the early stages of the invasion process. Clearly, results underline the wide spectrum of phenotypic effects of Wolbachia infection and, to our knowledge, Wolbachia infection of L. heterotoma appears to be one of the most virulent that has ever been observed in insects.

The elongation factor-1 alpha (EF-1α) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEα) that codes for a major part of the coding region of the EF-1α protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5′ and 3′ ends of the EF-1α gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5′ end of B. hominis C EF-1α were identified. A comparative study on the deduced amino acid sequences of BEα of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the speciesThe nucleotide sequence data reported in this paper are available in the GenBank database with the accession numbers as shown in Table 1.. A phylogenetic reconstruction analysis with other eukaryotic EF-1α sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.

Circulating anodic antigen (CAA) levels in serum and faecal egg counts are both quantitative measures of Schistosoma mansoni worm burdens. In this study, we have tested whether circulating anodic antigens can be included into an established egg count model. A data set with 3 repeated faecal egg count and serum CAA measurements of 50 individuals from a community in Burundi with moderate endemicity was used. By means of Monte Carlo simulation, both antigens and egg counts were related to an underlying worm pair distribution, taking into account the variation in repeated measurements (within individuals) and the variation in worm burdens (between individuals). Models with various assumptions (e.g. presence or absence of density-dependent egg production) were tested. Whereas observed and predicted egg counts agreed fairly well, the circulating antigen data could not be described satisfactorily. In particular, the predicted number of negative antigen concentrations was much lower than observed, while the number of light positives was overestimated. There seems to be a mechanism that causes a shift of expected (low) positive CAA concentrations towards zeros, which the proposed models do not provide for. Possible biological as well as assay-related mechanisms that may account for this shift are discussed. The assumption that serum CAA concentrations are a simple direct reflection of worm (pair) burdens could not be corroborated by this modelling exercise. Apparently, the relationship between (measured) CAA concentrations, egg counts and worm burdens in human S. mansoni infections is more complex than assumed.

The vectorial capacity of Biomphalaria pfeifferi from Ndiangue, Senegal, was investigated with an allopatric isolate of Schistosoma mansoni from Nkolbisson, Cameroon. The snail infection rate after exposure to a single miracidium per snail (MD1) was 56·3%, and 91·6% for snails exposed to 5 miracidia per snail (MD5). The minimum pre-patent period was 21 days. The mean total cercarial production for the MD1 group was 18511 cercariae per snail, and 9757 cercariae for the MD5 group. The maximum production of cercariae for 1 day was 4892 observed in a snail from the MD1 group at day 43 post-infection. The mean longevity of snails was higher in group MD1 (88 days p.i.) than in group MD5 (65 days p.i.). The chronobiological emergence pattern revealed a circadian rhythm with one shedding peak at mid-day. Comparisons are made with the vectorial capacity of the sympatric combination of B. pfeifferi Senegal/S. mansoni Senegal.

The sex ratio of adult worms has been observed biased towards males in Schistosoma mansoni under natural conditions. The origin of this bias is unknown. This paper determines whether males are more infective than females under controlled experimental bisexual conditions, and hence if the sex ratio is male-biased as a consequence of this. The male and female cercarial infectivities in uni- and bisexual vertebrate host infections using a range of controlled cercarial sex ratios were studied. The results showed that, in experimental unisexual infections, male cercariae were more infective than females, and that in experimental bisexual infections, male cercarial infectivity was similar to that of female, irrespective of cercarial sex ratio. Furthermore, cumulative male and female cercarial infectivity was maximal when sex ratio was equilibrated. The unbiased sex ratios obtained in our experimental bisexual infections are discussed in terms of behavioural and/or biochemical male–female interaction. Alternative explanations of the natural biased sex ratio are proposed.

Lactacystin, a specific inhibitor of proteasomes in eukaryotic cells, did not block parasite entry or the establishment of the parasitophorous vacuole, but did inhibit parasite growth and daughter cell budding, as well as DNA synthesis. Two other proteasome inhibitors, MG-132 and proteasome inhibitor 1, also blocked parasite growth and intracellular development. Adding lactacystin to established, dividing parasites, rapidly blocked parasite growth and daughter cell budding at all stages in the process. Pre-treating host cells with lactacystin did not block parasite entry or development. Moreover, under the conditions used, the host cells appeared not to be adversely affected indicating that host cell proteasome activity was not essential for parasite entry or development. Concomitant with these effects on parasite growth and division were morphological changes in the parasite including the appearance of whorls of ER-derived membranes presumably related to the failure to breakdown misfolded proteins. These changes were specific to lactacystin and were not seen in parasites treated with other protease inhibitors. Although the ER-derived structures resembled autophagic bodies, similar structures could not be induced by serum starvation nor did the membranous whorls acidify or undergo morphological changes consistent with autophagosomal maturation. These results highlight the possible role of proteasome activity in Toxoplasma in intracellular development and the regulation of parasite replication. However, how the dividing parasite recycles its organelles and the functional relationship between any lysosomal–autophagic pathway and proteasomes in the parasite remains unresolved.

The public health significance of hookworm disease is briefly reviewed. The latest evidence confirms that blood loss caused by the worms' feeding activity in the gut is a contributing factor in the development of poor iron status leading to iron-deficiency anaemia. The World Health Organization has identified adolescent girls and women of child-bearing age as high-risk groups regarding the impact of hookworm disease. The merits of treating pregnant women with anthelminthic drugs after the first trimester are discussed.

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup- like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

Different parasite species sharing the same intermediate host species may have similar or conflicting interests, depending on whether they are at the same stage in their life-cycle or whether they share the same definitive host. In the New Zealand cockle, Austrovenus stutchburyi, metacercariae of the digenean Meiogymnophallus sp. are positively associated with metacercariae of Curtuteria australis. This relationship is found in different cockle samples, and is independent of cockle shell size, which suggests that it is not merely the product of metacercariae accumulation over time. Both digenean species have the same definitive host, oystercatchers. Metacercariae of C. australis manipulate the phenotype of cockles, impairing the cockle's ability to burrow in the sediments. This makes the host more susceptible to oystercatcher predation. Thus Meiogymnophallus sp. can benefit by associating with C. australis and may hitch a ride with the manipulator parasite. This is supported by the finding that cockles impaired by C. australis and lying at the sediment surface harbour greater numbers of Meiogymnophallus than buried cockles. A third digenean species, whose sporocysts are found in cockles and which is not transmitted by predation, occurred only in surface cockles. Finally, a parasitic copepod with a direct life-cycle was found evenly distributed among buried and surface cockles, independently of their metacercarial loads. These results show that different parasite species do not use cockles in a random fashion, and that not all patterns of host use are consistent with shared or conflicting interests among parasites.

Franz cells (2-chambered, air/fluid phase static diffusion devices, previously used for the study of drugs across viable human skin) are utilized for the first time to investigate the process of infection of human skin by Schistosoma mansoni cercariae. Skin obtained from cosmetic surgery sources was used in the Franz cells to describe the temporal dynamics of the early interaction of cercariae with skin. At 38 °C, about 50% of cercariae applied in water to the epidermal surface of the skin were irreversibly attached within 1 min and after 5 min about 85% were similarly irrecoverable. The technique also provides the means of following the early penetration path of cercariae by histological methods. Franz cell results on the dynamics of attachment/early penetration have been compared with those obtained using artificial skin equivalents and non-human mammalian skin models in the context of the physical and chemical differences between these systems and viable human skin. It is concluded that Franz cells provide a convenient system for directly investigating the early phases of S. mansoni cercariae interaction with human skin.

In spite of their abundance, widespread distribution and medical importance, the phylogenetic relationships among Biomphalaria snails have received relatively little attention. We have collected and studied 29 populations of snails obtained from different localities from Brazil. We have sequenced the ribosomal DNA second internal transcribed spacer (ITS2) from the following Biomphalaria species: B. glabrata, B. tenagophila tenagophila, B. occidentalis, B. straminea, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and an outgroup species Helisoma duryi. The sequence from each species is unique. Three different methods of phylogenetic reconstruction were used (distance, maximum parsimony and maximum likelihood). The resulting phylogenetic trees obtained by these methods basically support current systematic relationships based on morphological characters alone. This study demonstrates that the ITS2 region contains markers useful for identification and determination of relationships among Biomphalaria species.

Numerous studies have shown that anthelminthic treatment can be effective in improving growth rates when given to malnourished children with ascariasis. Recent investigations have also indicated that Ascaris infections can affect mental processing in some school children. Poor socio-economic conditions are among the key factors linked with higher prevalences of ascariasis, as are defaecation practices, geophagia, cultural differences relating to personal and food hygiene, occupational necessity, agricultural factors, housing style, social class and gender. Chemotherapy is currently the major tool used for the strategic control of ascariasis as a short-term goal. In the long term, improvements in hygiene and sanitation are thought to aid long-term control considerably. Targeted treatment, especially when aimed at schoolchildren, has been a major focus of recent control efforts in some areas. Universal treatment reaches more people and thus decreases further aggregate morbidity, especially in nutritionally vulnerable preschool-age children. Selective treatment requires technical effort to identify heavily infected individuals; acceptance by the community may vary in less educated populations when some individuals receive treatment and others do not. Child-targeted treatment may be more cost-effective than population treatment in reducing the number of disease cases and, in high transmission areas, expanding coverage of a population can be a more cost-effective strategy than increasing the frequency of treatment.

A schistosome infection is initiated when the parasite penetrates the skin of a susceptible host. Relatively large quantities of protein are released by transforming cercariae compared to later larval stages. This represents the first parasite material to which the host's immune system is exposed, yet little is known about the proteins which are released during the first few hours post-transformation. We have shown that antiserum raised against such molecules was capable of imparting protection against a schistosome challenge infection upon passive transfer to naïve mice. By screening a cercarial cDNA library with this serum, 38 positive clones were identified. Sequence analysis showed these to represent 8 different molecules which included Schistosoma mansoni 21·7 kDa antigen, calcium-binding-protein and the vaccine candidate glutathione S-transferase (Sm28GST). In addition, 5 clones were isolated, 1 of which had significant homology to many cytochrome C proteins, another with leukocyte elastase inhibitors and 3 which represented novel molecules. Four clones were expressed in a prokaryotic high-level expression vector, sera produced against each purified recombinant protein and used subsequently to probe Western blots and parasite sections. The leukocyte elastase inhibitor homologue and 2 unknowns induced significant proliferation by lymph node cells recovered from mice vaccinated with irradiated cercariae. More strikingly, the 2 novel proteins stimulated very high levels of interferon γ (IFNγ) secretion both by lymph node cells and those recovered by broncho-alveolar lavage from the lungs of vaccinated mice. Such results will be discussed in the context of vaccine development.