East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five™ cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP[ratio ]p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are describedThe sequence data reported herein have been deposited in EMBL under Accession No. AJ422214. . In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

A Plasmodium falciparum single copy gene predicting a 122 kDa protein belonging to the M1 family of zinc-metallopeptidases was previously reported and related to erythrocytic schizont proteins of 96 (p96) and 68 (p68) kDa. By using protease inhibitors during parasite harvest and enzyme preparations, and polyclonal antibodies specific for 2 peptidic domains deduced from the gene, we identified the 120 kDa precursor and demonstrated its processing into p96 and p68. The N-terminal ends of p96 and p68 were mapped between glycine-123 and lysine-163, both proteins thus containing the catalytic domain. The purified enzyme, here named PfA-M1 (p96/p68), displayed strict aminopeptidase activity, optimal at pH 7·4, with broad substrate spectrum. Its inhibition and reactivation profiles were typical of zinc-metalloaminopeptidases. By Western blotting, PfA-M1 was detected in trophozoites, in addition to schizonts, but not in early rings. PfA-M1 was localized by indirect immunofluorescence confocal microscopy. In trophozoites, the labelling was diffuse in the parasite cytoplasm, with accumulations around the food vacuole. In schizonts, it turned progressively to a vesicle-like pattern, ending as a clear spot in released merozoites. The involvement of PfA-M1 in haemoglobin breakdown and erythrocyte reinvasion is discussed in light of the dual functions recently reported for several P. falciparum proteases.

The value of 2 PCR methods, targeting genomic and kinetoplast minicircle DNA respectively, was investigated for both diagnosis and prevalence studies of canine visceral leishmaniasis (CVL). The first method (R) was 5000-fold less sensitive than the second (method KRV). Both were tested for diagnosis of CVL in 44 sick dogs with confirmed disease using different biological samples. Method R was highly efficient when using invasive samples, but the use of method KRV proved necessary for a 100% sensitive diagnosis using peripheral blood. This method was applied to peripheral blood and skin samples in 263 dogs during a mass survey in the Cévennes focus. PCR was compared to serology and all results were analysed according to clinical status. The ‘CVL-infection’ prevalence was found to be 79.8% by PCR compared with 29.6% by serology: 89.4% of symptomatic and 65.2% of asymptomatic dogs harboured parasites in peripheral blood. This study confirms the high prevalence of asymptomatic carriers of Leishmania. In total, for the diagnosis of CVL in sick dogs, method R is recommended in view of its 100% positive predictive value (compared with 30% for method KRV). A strategy best adapted for prevalence surveys might combine serology and highly sensitive PCR on peripheral blood.

Plasmodium merozoites are covered by a complex coat of surface proteins. Several of the Merozoite Surface Proteins (MSPs) that make up this coat have been proposed as vaccine candidates although some of the MSPs are known to be highly polymorphic. We present here the first survey and analysis of the polymorphism in the recently characterized P. vivax surface protein PvMSP-3α. Full length or partial sequences were obtained for the Pvmsp-3α gene from isolates originating in Central and South America, Asia and the Pacific. The Pvmsp-3α sequence is remarkably diverse, but this extensive diversity is largely restricted to certain domains of the encoded protein. An acidic C-terminal domain and a smaller hydrophilic N-terminus are relatively conserved, while a central domain containing coiled-coil heptad repeats is highly polymorphic and in some isolates of P. vivax is partially deleted. Unlike other MSPs, there is no evidence of allelic families of PvMSP-3α gene sequences, and no evidence that certain patterns of polymorphism group within isolates of similar geographical origin. The distribution and nature of polymorphism suggest that there are functional restrictions on mutations in this gene, and have implications for inclusion of PvMSP-3α as a candidate in a P. vivax vaccine.

Although the mosquito vectors of malaria have an effective immune system capable of encapsulating many foreign particles, they rarely encapsulate malaria parasites in natural populations. A possible reason for this apparent paradox is that infection by malaria reduces the capability of the mosquito to mount an effective immune response. To investigate this possibility, we blood-fed Aedes aegypti mosquitoes on an uninfected chicken or on one infected with Plasmodium gallinaceum, and compared the proportions of the infected and uninfected mosquitoes that melanized a negatively charged Sephadex bead injected into the thorax 1, 2 and 4 days after blood-feeding. About 40% of the uninfected mosquitoes, but less than 25% of the infected ones, melanized the bead. The difference between infected and uninfected mosquitoes was most obvious 1 day after infection (at the parasite's ookinete stage), while the difference diminished during the early oocyst stage (2 days after infection) and disappeared at the later oocyst stage (4 days after infection). These results suggest that the parasite can either actively suppress its vector's immune response or that it modifies the blood of its chicken host in a way that reduces the efficacy of the mosquito's immune system. In either case, the reduction of immunocompetence can have important consequences for malaria control, in particular for the current effort being invested into the genetic manipulation of mosquitoes.

Polistes atrimandibularis is a species of obligate social wasp parasite, which introduces into nests of the host species P. biglumis, where it rears its own brood relying on the workers of the host species. Wasps of the host species discriminate between nestmates and non-nestmates by means of their epicuticular hydrocarbons. To overcome the host recognition systems, parasites have to modify their epicuticular recognition cues. Compared to individuals of the host species, parasites have 3–4 times less epicuticular hydrocarbons, although they share with their hosts the habitat and even the nests. We hypothesize that a dilution of the recognition cues in social parasites could have a facilitating role in entering host nests, in integrating within their host colony and in sneaking into secondary colonies to steal larvae and pupae.

Immune responses that kill pathogens or reduce their reproductive rate are generally important in protecting hosts from infection and disease. Pathogens that escape the full impact of such responses will survive, and any heritable genetic basis of this evasion will be selected. Due to the memory component of vertebrate immune responses, pathogens with rare alleles of a target antigen can have an advantage over those with common alleles, leading to the maintenance of a polymorphism. At the genetic level, there ought to be detectable signatures of balancing selection in the genes encoding these antigens. Here, methods for identifying these selective signatures are reviewed. Their practical utility for identifying which antigens are targets of protective immune responses is discussed.

Pulsed-field gel electrophoresis of intact chromosomes from 2 isolates of each of the 2 most pathogenic species of large Babesia parasites that infect dogs, i.e. Babesia canis (European species) and B. rossi (South African species), revealed 5 chromosomes in their haploid genome. The size of chromosomes 1–5 was found to be different in the 2 species, ranging from 0.8 to 6.0 Mbp. The genome size was estimated to be approximately 14.5 Mbp for B. canis and 16 Mbp for B. rossi, respectively. Within each species, the size of chromosomes 1–3 of B. canis and 1–2 of B. rossi was conserved between the 2 isolates, whereas the size of chromosomes 4–5 of B. canis and 3–5 of B. rossi was variable. Chromosomes 1–5 hybridized with a 28-mer telomeric oligonucleotide probe derived from Plasmodium berghei. When NotI-digested chromosomes of the 4 isolates were hybridized with the telomeric probe a maximum of 10 fragments was revealed. Moreover, hybridization of this telomeric probe to a Southern blot of genomic DNA from the 4 isolates, digested with a series of restriction enzymes, revealed a species-specific restriction map. Hybridization of intact or NotI-digested chromosomes of both species with 2 sets of 3 cDNA-antigen probes derived from each species, revealed no cross-hybridization between these B. canis and B. rossi genes.

The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3–25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.

Partial (DNA) sequences are presented for 2 nuclear (18S and 28S rRNA genes) and 2 mitochondrial (12S rRNA and ND1 genes) loci for 5 species belonging to the Schistosoma japonicum, S. sinensium and S. indicum groups of Asian Schistosoma. Fresh field isolates were collected and cultured for the following taxa: S. incognitum (S. indicum group, central Thailand), S. mekongi (S. japonicum group, southern Laos), S. ovuncatum (S. sinensium group, northern Thailand), S. spindale (S. indicum group, northeast Thailand and central Thailand isolates) and S. sinensium (S. sinensium group, Sichuan Province, China). This represents the first published DNA sequence data for S. ovuncatum and for S. sinensiums.s. from the type locality in China. The paper also presents the first sequence data at the above loci for S. incognitum (except for the 28S sequences) and S. sinensium. Congruence was observed between the phylogenies estimated for each locus, although the relationships of S. incognitum were not so well resolved. Fitch–Margoliash, maximum likelihood (ML) and maximum parsimony methods were used to estimate the phylogenies and the agreement between them was similar to that observed between loci. The ML tree was considered to best represent the data and additional 28S sequences (taken from the GenBank), for S. haematobium, S. japonicum, S. mansoni and Orientobilharzia turkestanicum, were used to construct an overall phylogeny. The S. indicum group taxa showed considerable divergence from the other Asian species and closest affinity with the African group. S. ovuncatum and S. sinensium appeared as sister taxa but their status as sibling species remained supported. The findings are discussed in the context of phylogeographical hypotheses for the origin of Schistosoma. An Asian origin for Schistosoma is also considered.

In order to improve our understanding of the host cell–parasite interactions in giardiasis, this study assessed the effects of Giardia lamblia on epithelial permeability and tight junctional ZO-1, determined whether epidermal growth factor (EGF) may affect Giardia-induced epithelial injury, and evaluated if EGF modulates epithelial colonization by live G. lamblia trophozoites. Permeability was assessed in assays of trans-epithelial fluxes of FITC-dextran, and ZO-1 integrity was characterized by confocal laser immunofluorescence microscopy in confluent epithelial cell monolayers. G. lamblia significantly increased paracellular permeability and disrupted tight-junctional ZO-1 of a novel non-transformed human small intestinal epithelial cell line (SCBN). Pre-treatment with EGF prevented the development of these abnormalities and significantly inhibited attachment of live trophozoites to the enterocytes, independently of a direct microbiocidal action. These findings demonstrate that G. lamblia may cause intestinal pathophysiology by disrupting tight junctional ZO-1 and increasing epithelial permeability. Apical administration of EGF prevents these abnormalities, and reduces epithelial colonization by the live parasites.

Due to shared characteristics with snail haemocytes, the Biomphalaria glabrata embryonic (Bge) cell line has been used as a model in vitro system for the study of snail–trematode interactions. In this study, Bge cells were used to characterize the adherence of snail host cells to schistosome primary sporocysts, and to test the effect of carbohydrates as inhibitors of this behaviour. Bge cells bound to the surface of >90% of Schistosoma mansoni sporocysts and, based on a semi-quantitative cell adhesion scale of 1–4, exhibited a cell adhesion index (CAI) of 2·45. This cellular adhesion was significantly inhibited in the presence of selected carbohydrate-containing substances. Fucoidan was the most potent inhibitor, reducing Bge cell-binding prevalence to approximately 50% and the CAI to 1·6. Other inhibitory compounds included mannose-6-phosphate, heparin, dextran sulfate, and various forms of the polysaccharide carrageenan. Fluoresceinated-fucoidan was found to attach to Bge cells confirming their ability to associate with sugar moieties. These results were further supported by the specific binding of surface biotinylated Bge cell proteins to sporocyst tegumental glycoproteins ranging from 40 to 120 kDa. N-linked tegumental carbohydrates appeared to represent ligands for Bge cell proteins since N-glycosidase treatment of blotted tegumental glycoproteins completely abolished biotinylated Bge protein binding. We hypothesize that the involvement of lectins as potential host cellular receptors in snail cell–sporocyst interactions, and suggest that negatively charged (mainly sulfated) carbohydrate moieties may represent the schistosome surface ligand(s).

During autumn 2000 an extensive cross-sectional survey of the prevalence of Schistosomiasis japonicum was conducted among about 4000 villagers within 20 villages in the Anning River Valley located in the southwestern Sichuan Province. Two procedures were used to assess infection status, the Kato-Katz thick smear procedure and a miracidia hatch test. Whereas the Kato-Katz procedure provides information on both prevalence and intensity, the hatch test provides only prevalence data, albeit on a much larger volume of stool. In addition, we performed Kato-Katz smears for 15 consecutive samples on a subset of 15 individuals. The proportion of both hatch-test and Kato-Katz positive individuals in the larger cross-sectional survey was 25%. The goal of the study was to estimate both the egg and worm distributions among risk groups using both the hatch and Kato-Katz tests from the cross-sectional data and the repeated Kato-Katz smears from the longitudinal data sets. As a prelude to parameter estimation, individuals were classified into risk groups by natural village and occupation; the proportion of Kato-Katz positive subjects among the risk groups varied from 10% to 60%. We used the statistical model of de Vlas et al. (1992) and Bayesian techniques to derive both estimates of and inference about the worm and egg distribution parameters. The parameter estimates imply (1) similar eggs per gram stool (e.p.g.) per worm pair compared with earlier estimates, (2) a range of worm burdens among the risk groups and (3) estimates of risk heterogeneity within groups is sensitive to prior information on the within-person variability in egg excretion.

Prevalence and abundance of Cryptosporodium parvum and Giardia spp. were studied in 3 species of rodents from forests and abandoned agricultural fields in N.E. Poland (Clethrionomys glareolus n = 459; Microtus arvalis n = 274; Apodemus flavicollis n = 209). Overall prevalence was consistently higher in the voles compared with A. flavicollis (70·6, 73·0 and 27·8% respectively for C. parvum and 93·9, 96·3 and 48·3% respectively for Giardia spp.). Prevalence and abundance of infection also varied markedly across 3 years with 1998 being a year of higher prevalence and abundance with both species. Fewer older animals (especially C. glareolus and M. arvalis) carried infection with C. parvum and infections in these animals were relatively milder. Although seasonal differences were significant, no consistent pattern of changes was apparent. Host sex did not influence prevalence or abundance of infection with C. parvum, but made a small contribution to a 4-way interaction (in 5-way ANOVA) with other factors in the case of Giardia spp. The 2 species co-occurred significantly and in animals carrying both parasites there was a highly significant positive correlation between abundance of infection with each, even with between-year, seasonal, host age, sex and species differences taken into account. Quantitative associations were confined to the 2 vole species in the study. These results are discussed in relation to the importance of wild rodents as reservoir hosts and sources of infection for local human communities.

The epidemiological role of the crab-eating fox Cerdocyon thous in the transmission of Leishmania infantum is assessed in a longitudinal study in Amazon Brazil. A total of 37 wild-caught foxes were immunologically and clinically monitored, and 26 foxes exposed to laboratory colonies of the sandfly vector Lutzomyia longipalpis, over a 15-month period. In total 78% (29/37) of foxes were seropositive for anti-Leishmania IgG on at least 1 occasion, and 38% (8/37) had infections confirmed by PCR and/or by culture. Point prevalences were 74% (serology), 15% (PCR), and 26% (culture). No signs of progressive disease were observed. None of the foxes were infectious to the 1469 sandflies dissected from 44 feeds. A conservative estimate of the possible contribution of foxes to transmission was 9% compared to 91% by sympatric domestic dogs. These results show that crab-eating fox populations do not maintain a transmission cycle independently of domestic dogs. The implication is that they are unlikely to introduce the parasite into Leishmania-free dog populations.

Perkinsus marinus is a protozoan responsible for dramatic mortality in the Eastern oyster, Crassostrea virginica, but not in the Pacific oyster, C. gigas. To understand the host–parasite relationship, we inoculated P. marinus trophozoïtes into the shell cavity of C. gigas and measured, over 2 months, (i) intensity of infection, (ii) protease inhibitory activities against P. marinus proteases and against bovine α-chymotrypsin, (iii) plasma haemagglutinin titre, (iv) plasma protein concentration, (v) plasma lysozyme activity and (vi) total haemocyte count. We observed that the highest protease inhibitory activities and haemagglutinin titres (3–10 days post-challenge) preceded parasite elimination (initiated 7 days post-challenge). In contrast, plasma protein concentration, lysozyme activity and total haemocyte count showed no significant modification following the challenge. It is hypothesized that the capacity of C. gigas to increase its protease inhibitors represents the key event in resistance to parasite infection by neutralizing the proteases secreted by P. marinus, thus preserving the oyster haemagglutinins from degradation. Such haemagglutinins will be ready to act as opsonins stimulating phagocytosis of parasites.

Intra-clonal phenotypic (antigenic) variation is used by many pathogens to evade the consequences of immune-mediated killing by mammalian hosts. In this substantially theoretical article, I emphasise that antigenic variation (sensu stricto) involves no change in genotype; its importance as a mechanism for promoting pathogen transmission and its polyphyletic origin. From a functional perspective, antigenic variation is constrained by the requirement to meet five conditions. These are: capability to express several antigens against which functional immunity predominates; capability to interact with the environment; mutually exclusive expression of variable antigens in each cell within an infection; mutually exclusive expression in the within-host pathogen population and the capability for population growth within a host. Meeting these conditions leads to chronicity of infection and high rates of hierarchical and reversible switching of expression between variable antigens. The organisation of hierarchical expression is discussed in some detail.