The three trypanosomatid genome projects have employed common strategies which include: analysis of pulsed-field gel electrophoretic chromosomal karyotypes; physical mapping using big DNA (cosmid, pacmid P1, bacterial artificial chromosome, yeast artificial chromosome) libraries; partial cDNA sequence analysis to develop sets of expressed sequence tags (ESTs) for gene discovery and use as markers in physical mapping; genomic sequencing; dissemination of information through development of web-sites and ACeDB-based fully integrated databases; and establishment of functional genomics programmes to maximize useful application of genome data. Highlights of the projects to date have been the demonstration that, despite extensive chromosomal size polymorphisms for diploid homologues within Africa trypanosomes, T. cruzi or Leishmania, the physical linkage groups for markers on each chromosome are retained across all isolates/species studied within each group. For African trypanosomes, detailed analysis of chromosome 1 has demonstrated that repetitive sequences and the two retroposon-like elements RIME and INGI are localized to a defined region at one end of the chromosome, with the bulk of the central region of the chromosome containing genes coding for expressed proteins. Comparative mapping shows that, although subtelomeric changes account for a large proportion of the polymorphism in chromosome size in African trypanosomes, there are significant expansions and contractions in regions across the entire chromosome. The highlight of the genomic sequencing projects has been the demonstration of just 2 putative transcriptional units of chromosome 1 of Leishmania major, extending on opposite strands from a point in the central region of the chromosome. A similar observation made on 93·4 kb of contiguous sequence for T. cruzi chromosome 3 suggests the presence of promoter and regulatory elements at the junctions of large polycistronic transcriptional units. All data obtained from the genome projects are made available through the public domain, which has prompted changing philosophies in how we approach analysis of the biology of these organisms, and strategies that we can employ now in the search for new therapies and vaccines.

Binding to the midgut microvillar surface in the sandfly Phlebotomus papatasi is a prerequisite for successful development of Leishmania major within the gut of the vector. This paper describes a method for detecting microvillar-associated proteins which act as ligands for the parasite surface glycoconjugate lipophosphoglycan (LPG). Adhesion of LPG to midgut proteins was visualized by probing midgut extracts with LPG using a Western ligand blotting technique. Procyclic L. major LPG bound to a microvillar polypeptide band of 65 kDa (estimated in the non-reduced state) and bound variably to several lower molecular weight bands, probably degradation products or subunits of the primary binding polypeptides. Specificity of binding was confirmed by co-incubating biotinylated LPG with an LPG-specific mAb which resulted in a great reduction in binding.

Polysporoplasma sparis infection was studied in gilthead sea bream from different mariculture systems of the Spanish coasts. Culture conditions influenced the infection dynamics, as the parasite appeared only in semi-intensive cultures and was not found in intensive closed systems nor in open ones. No clear seasonal pattern was observed. No fish weighing less than 51 g was found parasitized in any group. A statistically significant dependence between infection prevalence and host weight was observed in some growing stocks. Light and transmission electron microscope observations revealed serious damage in the trunk kidney. Glomerular disease was provoked by the progressive occupation of the glomerular capillaries by P. sparis spores. Tubular epithelial cells were also affected. Inflammatory responses appeared towards the end of the infection, and consisted mainly of melanomacrophages and eosinophils. Rodlet cells were common close to infected capillaries and debris of rodlet sacs formed a belt encircling capillary vessels. Cytochemistry demonstrated the lipidic nature of these sacs and the glycogen and glycoprotein composition of the cytoplasmic granules of rodlet cells.

The aim of this investigation was to examine whether macrophage and Leishmania major glutathione were involved in either host or parasite protection against NO cytotoxicity. Buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl-cysteine synthase, caused a complete and irreversible depletion of macrophage glutathione, but only a 20% and reversible decrease in L. major glutathione. Glutathione-depleted macrophages, when activated with IFN-γ/LPS, released less than 60% of the NO produced by untreated macrophages, resulting in a corresponding decrease in their leishmanicidal activity. BSO-treated macrophages were more susceptible to the cytotoxic effects of the NO donor SNAP. Treatment of macrophages with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase and trypanothione reductase or with Br-Octane, a glutathione-S-transferase substrate, resulted in a transient decrease in glutathione levels and did not increase the susceptibility of the macrophages to SNAP. Treatment of the promastigote forms of L. major with BCNU resulted in an 80% decrease in total glutathione concentration with no concomitant change in viability. However, this treatment rendered the parasites more susceptible to SNAP. Finally, macrophage glutathione protected the internalized L. major from SNAP. Overall, these results demonstrate that glutathione is an essential protective component against NO cytotoxicity on both macrophages and parasites.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.

A number of genomes of parasitic organisms are presently being sequenced in the public domain, including Plasmodium falciparum, Leishmania major and Trypanosoma brucei with the likelihood of at least expressed sequence tag (EST) projects for several filarial and apicomplexan species. The early and timely release of sequence data to the community via the World Wide Web (www), and the public databases, (EMBL and GENBANK), forms an invaluable resource. Data mining, or ‘haystack searching’ this resource is becoming more fruitful to all members of the scientific community as the volume of data, diversity of genomes sampled, and accessibility increase.

Using an age-fractionated RBC model, we investigated the in vitro sensitivity of artemether in β-thalassaemic RBC infected with the K1 and FC27 strains of Plasmodium falciparum and, to study the role of oxidant stress in modulating the sensitivity pattern, pro-oxidant (riboflavin) and antioxidant (vitamin E) agents were added to cultures in the presence of artemether. With the FC27 strain, the artemether IC50 doses in thalassaemic samples (whole blood and fractions) were significantly higher compared to equivalent normal RBC samples (P<0·05). However, with the K1 strain, such a significant difference was not demonstrable. The addition of vitamin E reduced the antimalarial effect of artemether in both the FC27 and K1 strains (P<0·0001). In contrast, the addition of riboflavin resulted in a significant increase in antimalarial activity (P<0·0001). This effect of the drug combinations was not influenced by the red cell type (P<0·0001) and there was no interaction between red cell type and drug type (P<0·0001). These findings show that reduced sensitivity to artemether occurs in whole blood and age-fractionated β-thalassaemic trait RBC. It appears that the RBC redox status does not influence the sensitivity to artemether.

We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed.

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.

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.

Community data on the prevalence of helminth infections is important for guiding health policy, but expensive to collect. As a result most surveys focus on school-aged children, frequently using schools as a sentinel population. Since there already exists a vast amount of data on infection levels in school-aged children, but limited community-based data, we undertook a literature search on age-stratified infection data for intestinal nematode infections and schistosomiasis in Africa, to investigate whether estimates of the prevalence of infection in school-aged children could provide an index for determining community prevalence. The observed data on prevalence of infection in infants, school-aged children and adults were fitted using linear and logistic regression models which take into account variation in sample prevalences. Despite the wide variation in study sites, the observed relationship between community prevalence and school-aged prevalence was remarkably consistent for each parasite species. The prevalence of infection in school-aged children alone was shown to be higher than the predicted prevalence in the community, but the degree of overestimation was dependent on the parasite species and the level of infection. The results suggest that the prevalence of infection in school-aged children could provide a cost-effective predictive tool which can be used at a district/national level to identify target areas for control and to evaluate the numbers at risk of infection.

Cysts of Blastocystis ratti were produced in vitro by culturing the parasite in Iscove's modified Dulbecco's medium (IMDM) with increasing concentrations of horse serum. Yields up to 3×106 cysts/ml of culture medium were obtained after 72 h. Encystation efficiency was time, strain and inoculum size dependent. A viability of >70% was determined by flow cytometry employing fluorescein diacetate and propidium iodide staining. The presence of chitin as a cyst wall component was demonstrated by Calcofluor White M2R staining with which cystic stages showed blue fluorescence. The changes in morphology during excystation were examined by transmission electron microscopy. The cyst enlarged in size and some vacuoles appeared within the condensed cytoplasm. The vacuoles were full of inclusions and small glycogen aggregates. Coalescence of the vacuoles led to central body formation. Glycogen deposits were prominent throughout the excystation process. Some cysts divided by binary fission before the completion of the excystation.

Genome projects for the parasitic helminths Brugia malayi (a representative filarial nematode) and Schistosoma were initiated in 1995 by the World Health Organization with the ultimate objectives of identifying new vaccine candidates and drug targets and of developing low resolution genome maps. Because no genetic maps are available, and very few genes have been characterized from either parasite group, the first goal of both Initiatives has been to catalogue new genes for future placement on chromosome and physical maps. These genes have been identified by the expressed sequence tag (EST) approach, utilising cDNA libraries constructed from diverse life cycle stages. To date, the Initiatives have deposited over 16000 Brugia ESTs and nearly 8000 Schistosoma ESTs in Genbank's dbEST database, corresponding to 6000 and over 3600 genes respectively (33% of Brugia's estimated gene compliment, 18–24% of that of Schistosoma). Large fragment, genomic libraries have been constructed in BAC and YAC vectors for studies of genomic organization and for physical and chromosome mapping, and public, hypertext genomic databases have been established to facilitate data access. We present a summary of progress within the helminth genome initiatives and give several examples of important gene discoveries and future applications of these data.

This study deals with the ecology of Lyme borreliosis in Europe. The relationships between Borrelia burgdorferi sensu lato spirochetes, Clethrionomys and Apodemus rodent reservoirs and the Ixodes ricinus tick vector were investigated during 16 consecutive months in an enzootic area in Switzerland. Cultivation of ear skin biopsies was used to isolate spirochetes from C. glareolus, A. sylvaticus, A. flavicollis and Glis glis. Borrelia infection was more frequently observed in Clethrionomys than in Apodemus. Tick xenodiagnosis was used to determine the infectivity of rodents. The infection rate in ticks fed on Clethrionomys was higher than that in ticks fed on Apodemus, but Apodemus yielded more infected ticks than Clethrionomys because of a better tick moulting success. Xenodiagnostic ticks were placed into BSK medium to obtain isolates. Isolates from rodents and rodent-feeding ticks were all identified as B. afzelii. The follow-up of the infectivity status of repeatedly recaptured rodents clearly demonstrated that these hosts remained infective for ticks during winter till the following spring. Comparing C. glareolus and A. sylvaticus, each rodent species showed different host infection, different host infectivity and contributed differently to the moulting success of feeding ticks. These factors influence differentially the pattern of transmission of B. afzelii from Clethrionomys voles and Apodemus mice to I. ricinus ticks.

American trypanosamiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection in natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, the mini-exon and the 24Sα rRNA gene loci have shown that T. cruzi stocks can be clustered in 2 major phylogenetic groups: lineage 1 and lineage 2. In this report, 68 recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical areas in Rio de Janeiro, Brazil, have been typed based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, 26 stocks were from golden-lion tamarins, 31 from opossums, 2 from rodents and 1 from a three-toed sloth. Thirty (44% – 30/68) isolates were typed as lineage 1, while 36 (53% – 36/68) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, 3% (2/68) of the isolates were typed as lineage 1+lineage 2. Using these geographical regions as models of sylvatic environments, it was observed that 96% of the Didelphis marsupialis were infected by lineage 2 isolates, while all 26 golden-lion tamarins were infected by lineage 1. The results show preferential association of the 2 lineages of T. cruzi with different hosts, composing the complexity of the sylvatic cycle.

We investigated the segregation of an intracellular microsporidian parasite during host cell division. A time-course experiment was carried out to examine the distribution of parasites relative to host chromosomal DNA via light and electron microscopy. Fluorescent light microscopy and EM studies showed that the parasite lay in the perinuclear zone of the host cell during interphase and segregated to daughter cells at mitosis. At metaphase, the parasite was frequently closely associated with host microtubules and mitochondria. Electron-dense bridges were observed between the parasites and the host microtubules and also between host mitochondria and microtubules. The study suggests that both the parasite and the host cell organelles segregate in association with spindle microtubules.

The formation and development of monogenean communities on the gills of roach fry was followed in 1992 from early June to October (size range 9 to 47 mm). Roach fry (n=291) were sampled weekly from the small, humic River Rutajoki in central Finland. A further 209 roach fry were reared in a fish farm supplied by water from the river. Four Dactylogyrus species were found: D. nanus, D. crucifer, D. micracanthus and D. suecicus. Other species found on the gills were Gyrodactylus sp. and Paradiplozoon homoion. The first Dactylogyrus juvenile occurred on a 12 mm long fish fry in late June and the first adult (D. nanus) 1 week later in Tank 1. D. nanus was also the most common parasite in the river. Young fry had high numbers of Gyrodactylus sp. on the gills compared with adult roach in previous studies. Signs of site preference were found; D. nanus and D. micracanthus preferred the 2nd gill-arch and Dactylogyrus juveniles the outmost one. Abundances of monogenean infracommunities increased until the middle of August when, on average, 1·9 species and 3·7 worms per fry were noted and decreased then as the water temperatures fell. A lack of competition between species is suggested; for example, D. crucifer appeared on the gills at that time when abundance of D. nanus was at its highest. The roach fry hatched and reared in the fish farm revealed that larvae of the most common dactylogyrid species were able to disseminate and colonize over 120 m from the river to the farm. Influence of change colonization events was shown by the appearance and development of Gyrodactylus sp. population on roach fry in only 1 of the 2 tanks at the farm.

This study compared the biological and genetic properties of a bovine (NC-SweB1) and a canine (NC-Liverpool) isolate of Neospora caninum. A mouse model for CNS infection demonstrated marked differences in pathogenicity between the isolates. NC-Liverpool induced severe clinical signs of neosporosis in 57/58 mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss. In contrast NC-SweB1 induced similar but less severe symptoms in a much smaller proportion of mice over the same time-period. Statistically significant differences were observed between the isolates in the response (mean weight loss) of mice through time to the different doses inoculated. Histopathological effects on brain tissue reflected the isolate-based differences described above. NC-Liverpool infection resulted in intense inflammatory infiltrates and highly necrotic lesions whereas NC-SweB1 induced a milder meningoencephalitis. Passage in cell-culture over a period of 14 months did not affect the pathogenicity of NC-Liverpool. Immunoblots showed that antibodies to N. caninum appeared earlier in mice inoculated with NC-Liverpool than with NC-SweB1. Finally, RAPD–PCR analysis of NC-Liverpool DNA generated profiles distinct from that observed with DNA from NC-SweB1 or Toxoplasma gondii. In summary this study provides evidence for significant biological and genetic differences between 2 isolates of N. caninum.

We have previously shown that oral treatment with the leaf extract of the plant Kalanchoe pinnata (Kp) significantly decreases the lesion size and the parasite load in BALB/c mice infected with Leishmania amazonensis. Here we report on the mode of action of Kp, particularly on the induction of nitric oxide (NO) production by macrophages. We observed that Kp has no direct inhibitory activity on extracellular promastigotes, but effectively decreases the intracellular amastigote growth in a dose-related fashion. A 58% reduction in amastigote growth induced by 500 μg/ml Kp was associated with a 6-fold increase in the production of NO by the macrophages. IFN-γ synergistically enhanced the NO-stimulating effect of Kp in culture. Co-treatment with the inducible NO synthase enzyme inhibitor l-NG-monomethyl-arginine abolished the antileishmanial effect of Kp in vitro and in L. amazonensis-infected BALB/c mice. These results indicate that the protective effect of Kp in leishmaniasis may not be due to a direct effect on the parasite itself but rather to activation of the reactive nitrogen intermediates pathway of macrophages.