Epitope mapping of a murine monoclonal antibody (mAb), 5H12, prepared against live Plasmodium falciparum-intected red blood cells indicated that the epitope consisted of amino acid residues 474–487 of the human anion transport protein, band 3. mAb 5H12 enhanced cytoadherence, but inhibited the CD36-like mediated resetting. A synthetic peptide based on the sequence of the epitope (FSFCETNGLE) blocked both resetting and cytoadherence, suggesting that this amino acid sequence may form the CD36-like receptor. The CD36-like region of band 3 was antigenically distinct from platelet or endothelial CD36.

Theileria sergenti Shintoku stock consists of 2 parasite populations bearing 2 atlelic forms of p33/32, an immunodominant piroplasm surface protein. Parasite population changes during parasite passages among cattle and tick vectors, and during persistent infection in individual calves were analysed by using allele-specific polymerase chain reaction (PCR). The parasite DNAs were prepared from piroplasms from calves which had been infected with Shintoku stock by inoculation of sporozoite stabilates or parasitized erythrocytes, and from sporozoite stabilates which had been prepared from Shintoku stock-infected ticks. Changes in a dominant parasite population were demonstrated during transmission from calves to vector ticks and from infected ticks to calves. Parasite population changes were also apparent during persistent infection in cattle over several months, and this change is thought to occur under host immune pressure. The results of this study indicate that expression of diverse forms of p33/32 may play a role in parasite persistence within mammalian hosts and its transmission from tick vector.

Codon usage of Crytosporidium parvum was compared with those of other Eimeriorina Toxoplasma gondii and Eimeria tenella and revealed a biased use of synonymous codons with a preference for NNU (40·0%) and NNA (33·4%). There was no close resemblance of the codon usage of C. parvum to T. gondii (correlation coefficient, r = 0·14) or E. tenella (r = 0·14) but it was similar to Entamoeba histolytica (r = 0·75) and Plasmodium falciparum (r = 0·5). Analysis of the codon usage in homologous gene sequences (actin, β-tubulin) also failed to reveal a close relationship between C. parvum and T. gondii or E. tenella. The low usage codons in C. parvum were most frequently used codons in T. gondii and E. tenella. These observations are consistent with 18S rRNA sequence analysis which shows no close relationship of Cryptosporidium with other Eimeriorina (Sarcocystis, Toxoplasma and Eimeria) and questions the validity of the current classification of C. parvum.

Host immunity will result in increased homozygosity at immunogenic loci if they encode products that elicit allele-specific immune responses. This increased homozygosity can be detected by comparison to ‘neutral’ (i. e. unselected) loci in the same genome which act as controls for the various epidemiological factors, such as biting rate, which affect homozygosity. Numerical results suggest that homozygosity should be 20 to 500 % higher at immunogenic loci, a result which is robust to changes in host death rate and the degree of linkage between the immunogenic and neutral loci. The same logic applies to loci which encode drug resistance: treated individuals with resistant infections will transmit zygotes homozygous for the resistance allele. It is argued that this increased homozygosity at putative immunogenic loci can act as a diagnostic feature to test using field data. The method also serves as a potentially very powerful method of identifying immunogenic and drug-resistant loci in laboratory studies of species such as the murine malaria Plasmodium chabaudi.

We have investigated the metabolic disturbances in 2 murine models of blood-stage malaria, Plasmodium chabaudi and Plasmodium yoelii. Blood glucose, plasma insulin and parasitaemia were measured in normal and infected mice before and after treatment with diazoxide, adrenaline, Sandostatin and quinine. Severe hypoglycaemia and marked hypersinsulinaemia developed during both infections. A single injection of diazoxide (25 mg/kg i.p.) or adrenaline (0·03 mg s.c.) lowered insulin concentrations in normal mice, reversed the hypoglycaemia in both infections and significantly reduced the hyperinsulinaemia in P. chabaudi-infected mice (P < 0·0001). Higher doses of Sandostatin (500 μg/kg s.c.) were required to reverse hypoglycaemia. Quinine (25 mg/kg i.p.) significantly increased blood glucose in normal and infected mice (P < 0·001) and no hypoglycaemia was observed in mice with normal blood glucose for more than 3 h. This study shows that the major cause of hypoglycaemia in murine malaria is hyperinsulinaemia rather than high consumption of glucose by host and parasites or chemotherapy with quinine, and that hypoglycaemia can be reversed by correcting the hyperinsulinaemia.

Samples of DNA from a panel of Giardia isolated from humans and animals in Europe and shown previously to consist of 2 major genotypes–‘Polish’ and ‘Belgian’–have been compared with human-derived Australian isolates chosen to represent distinct genotypes (genetic groups I–IV) defined previously by allozymic analysis. Homologous 0·52 kilobase (kb) segments of 2 trophozoite surface protein genes (tsa417 and tsp11, both present in isolates belonging to genetic groups I and II) and a 1·2 kb segment of the glutamate dehydrogenase (gdh) gene were amplified by the polymerase chain reaction (PCR) and examined for restriction fragment length polymorphisms (RFLPs). Of 21 ‘Polish’ isolates that were tested, all yielded tsa417-like and tsp11-like PCR products that are characteristic of genetic groups I or II (15 and 6 isolates respectively) in a distinct assemblage of G. intestinalis from Australia (Assemblage A). Conversely, most of the 19 ‘Belgian’ isolates resembled a second assemblage of genotypes defined in Australia (Assemblage B) which contains genetic groups III and IV. RFLP analysis of gdh amplification products showed also that ‘Polish’ isolates-were equivalent to Australian Assemblage A isolates (this analysis does not distinguish between genetic groups I and II) and that ‘Belgian’ isolates were equivalent to Australian Assemblage B isolates. Comparison of nucleotide sequences determined for a 690 base-pair portion of the gdh PCR products revealed ≥ 99·0% identity between group I and group II (Assemblage A/‘Polish’) genotypes, 88·3–89·7% identity between Assemblage A and Assemblage B genotypes, and ≥ 98·4% identity between various Assemblage B/‘Belgian’ genotypes. The results confirm that the G. duodenalis isolates examined in this study (inclusive of G. intestinalis from humans) can be divided into 2 major genetic clusters: Assemblage A (= ‘Polish’ genotype) containing allozymically defined groups I and II, and Assemblage B (= ‘Belgian’ genotype) containing allozymically defined groups III and IV and other related genotypes.

We have examined genetic variability in parasites in the context of ecological interactions with the host. Recent research on Echinococcus, Giardia and Cryptosporidium has been used to illustrate: (i) the problems that parasite variability and species recognition pose for understanding the complex and often controversial relationship between parasite and host occurrence; (ii) the need for accurate parasite characterization and the application of appropriate molecular techniques to studies on parasite transmission if fundamental questions about zoonotic relationships and risk factors are to be answered; (iii) our lack of understanding about within-host interactions between genetically heterogeneous parasites at the inter-and intraspecific levels, and the significance of such interactions with respect to evolutionary considerations and the clinical outcome of parasite infections. If advances in molecular biology and mathematical ecology are to be realized, we need to give serious consideration to the development of appropriate species concepts and in vivo systems for testing the predictions and assumptions of theoretical models.

In order to study the effects of acclimatization of Plasmodium in β-thalassaemic mice, we used a mouse model of β-thalassaemia (DBA/2J/β-thal/β-thal), similar to that observed in humans. We acclimatized 3 rodent malarias (P. berghei, P. chabaudi and P. yoelii) in DBA/2J and DBA/2J/β-thal/β-thal mice lines, by 4 intraperitoneal serial transfers. All 3 rodent malarias developed in red blood cells of β-thalassaemic mice without losing their virulence. There was no delay in infection and peaks of parasitaemia were similar in β-thalassaemic and normal mice. The mortality occurred earlier in β-thalassaemic mice than in control mice for P. berghei and P. chabaudi. This difference was more pronounced for P. yoelii NS where normal mice did not die. These results could be explained by a failure of erythropoiesis in β-thalassaemic mice, which are unable to compensate for the destruction of red blood cells by the parasites, and the mice died of anaemia. Ultrastructural examination of the rodent malaria parasites in β-thalassaemic RBC showed a normal development even in the presence of Heinz bodies. In conclusion, no effective protection against malaria was provided by the β-thalassaemia in this mouse model.

Recent work on the inheritance of susceptibility to malaria suggests that, over what may have been a relatively short evolutionary period, a remarkably diverse series of gene families have been modified in response to the selective drive of this single infection. The phenotype consequences are not confined to the red cell, but involve the immune system, cytokines and many other systems. It seems likely that the mechanisms of variation in genetic susceptibility to other infective agents will reflect at least a similar degree of complexity and, if the selective pressures have been present for longer periods of our evolutionary history, may be even more diverse. This may have important implications for work directed at trying to define susceptibility loci for current infectious and non-infectious diseases.

Since 1993, strains of Entamoeba histolytica sensn lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Sialic acid on the red cell surface plays a major role in invasion by the malaria parasite Plasmodium falciparum. The NeuAc(α2,3) Gal motif on the O-linked tetrasaccharides of the red cell glycophorins is a recognition site for the parasite erythrocyte-binding antigen (EBA-175). Consequently, the interaction of P. falciparum and the red cell might share homology with that of the influenza virus. The cellular interactions of P. falciparum were examined for their sensitivity to 4-guanidino-2,3-didehydro-D-N-acetyl neuraminic acid (4-guanidino Neu5Ac2en), a potent inhibitor of influenza virus sialidase. Parasite invasion and subsequent development was unaffected by the sialidase inhibitor. The inhibitor did not affect rosette formation of parasite-infected erythrocytes with uninfected cells nor their cytoadherence to C32 melanoma cells. Furthermore, we were unable to confirm the presence of a previously reported parasite sialidase using sensitive fluorometric or haemagglutination assays, neither was any malarial trans-sialidase identified. We conclude that P. falciparum possesses neither sialidase nor trans-sialidase activity and that an inhibitor of influenza virus sialidase has no effect on important cellular interactions of this parasite.

Iron overload in BALB/c mice by treatment with ferric ammonium citrate promotes the hepatic development of Plasmodium yoelii in vivo and in vitro. This was the result of increased penetration of the parasite into hepatocytes since no effect was observed on parasite transformation or maturation. These results could explain why in endemic regions iron supplementation led, in certain studies, to an increase in clinical episodes of malaria and in the prevalence of malaria infection.

Two genetically defined parents of Eimeria tenella, each characterized by a selectable phenotype, were mated to provide ‘recombinant’ oocysts for an analysis of the inheritance of DNA markers into the sporocyst stage of the life-cycle. A total of 23 populations was established from infection with a putative single sporocyst and their inheritance of DNA markers provided compelling evidence that the 2 sporozoites within each sporocyst are genetically identical. Each of the 5 polymorphic DNA markers used segregated independently within the ‘recombinant’ parasites and only 1 of the parental forms was inherited in 22 of the populations aimed to have been established from a single sporocyst.

Ex vivo culture of spleen cells from BALB/c mice infected with 2 × 106Leishmania major (L.major) promastigotes were cultured with ConcanavalinA (ConA) or leishmanial antigen (L. Ag) and tested for prostaglandin E2 (PGE2) and for leukotriene B4 (LTB4), in order to study their involvement in the evolution of cutaneous leishmaniasis and the connexion with lymphokine-mediated responses. The data were compared with those obtained in BALB/c mice protected against L. major by sublethal irradiation (550 rad; cured mice). In the unprotected BALB/c mice the levels of PGE2 that were responsible for the depression of interferon-γ (IFN-γ) and tumour necrosis factor-α (TNFα) Th1-associated cytokines and for the relative increase in the interleukin-4 (IL-4) became higher and higher as the lesion progressed. On the contrary, the cured mice produced levels of PGE2 similar to normal uninfected controls, high levels of TNFα and IFN-γ and low levels of IL-4. Elevated levels of LTB4 were detected in the early stage of infection in the unprotected mice compared to cured ones, a sign of more intense inflammation and a stimulus for the recruitment of inflammatory cells. The observation that exogenous LTB4 was able to enhance in vitro both Th1 cytokines in cured mice and Th2 cytokines in unprotected ones suggests that LTB4 could act in the recruitment of the T cells already committed to Th1 or Th2 phenotype.

Soluble adult worm antigens (SWAP) of Schistosoma mansoni were fractionated by fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that may elicit cell responses in human schistosomiasis. SWAP fractions were eluted by 20 HIM Tris–HCl solution (pH 9·6) with an increasing gradient of 1 M NaCl. The FPLC system was able to resolve 6 fractions, enumerated I to VI, according to the NaCl gradient. The analysis of each fraction on SDS–PAGE showed that fractions I to IV were constituted by multiple protein bands with Mr ranging from 21 to > 200 kDa. Large amounts of nucleic acids were evidenced in fractions V and VI, as revealed by ethidium bromide staining of agarose electrophoresis gels. Using ELISA, it was shown that sera from chronic schistosomiasis patients contained antibodies that recognized antigens in practically all fractions. Studies were designed to investigate the capacity of these fractions to induce cell proliferation and granuloma formation. It was demonstrated that fraction III stimulated a significant proliferative response of peripheral blood mononuclear cells (PBMC) from chronic schistosomiasis patients. However, fraction III coupled to polyacrylamide beads induced small granuloma formation in vitro, whereas beads coated with fractions I, II and V were able to induce significant granuloma reactions.

Many parasites that use intermediate hosts are transmitted to the next host through predation. If the next host's fitness is strongly reduced by the parasite, it is under selection either to recognize and avoid infected intermediate hosts or to exclude that prey species from its diet when alternative prey are available. We investigated the predator-prey interaction between laboratory bred three-spined sticklebacks (Gasterosteus aculeatus), the second intermediate host of the cestode Schistocephalus solidus, from 2 parasitized and 1 unparasitized population, and different prey types: infected and uninfected copepods and size-matched Daphnia as alternative prey. Copepods with infective procercoids were more active, had a lower swimming ability and were easier to catch than uninfected controls. The sticklebacks preferred moving copepods. Therefore parasitized copepods were preferentially attacked and consumed. There was no effect of the sticklebacks' parent population being parasitized or not. The sticklebacks switched from Daphnia to (uninfected) copepods in the course of a hunting sequence; this switch occurred earlier in smaller fish. With this strategy the fish maximized their feeding rate: Daphnia were easier to catch than copepods but increasingly difficult to swallow when the stomach was filling up especially for smaller fish. However, there was no indication that sticklebacks from infected populations either consumed Daphnia rather than copepods or switched later in the hunting sequence to consuming copepods than fish from an uninfected population. Thus, sticklebacks did not avoid parasitized prey although S. solidus usually has a high prevalence and causes a strong fitness reduction in its stickleback host.

A Leishmania infantum cDNA library was screened with sera from dogs with viscero-cutaneous leishmaniasis. Sequence analysis of a positive clone isolated from the library revealed that it coded for the carboxyl-terminal region of a member of the 70-kDa heat-shock protein family. The full-length sequence of the L. infantum hsp70 gene was determined after isolation of genomic clones. This protein shows a high degree of sequence conservation with the homologous protein from other organisms. To test its antigenicity a recombinant Hsp70 protein fused to the maltose-binding protein was produced in Escherichia coli using the expression vector pMAL-cRI. By FAST-ELISA assays it was observed that while the complete recombinant protein was recognized by 100% of the sera, the 20 carboxyl-terminal amino acids of the protein were only recognized by 30% of those sera. Thus, although a B-cell epitope must be present within the carboxyl terminal end of the protein other antigenic determinant(s) must reside out of this region. The analysis of the cross-reactivity with mouse Hsp70 by Western blotting strongly suggests that the anti-Hsp70 antibodies generated by infection with L. infantum are directed at specific determinants of the L. infantum Hsp70. Thus, our results indicate that anti-Hsp70 autoantibodies are not induced during Leishmania infection.

We have extracted a protein of 14 kDa from purified oocyst walls of several Eimeria species. Polyclonal antibodies were raised in rats against the 14 kDa proteins of E. acervulina and E. tenella. On immunoblots these antisera reacted in a highly specific manner with the homologous 14 kDa antigens, but not with heterologous antigens. In addition, specific binding of the two antisera to oocyst wall fragments of E. acervulina and E. tenella was demonstrated by immunofluorescence. Partial amino-terminal sequences comprising 20 amino acid residues were obtained from the 14 kDa oocyst wall proteins of E. acervulina and E. tenella. They are characterized by an abundance of amino acids containing hydroxyl groups in their side chains (serine, tyrosine, threonine). Binding of the oocyst wall protein of E. tenella by peanut agglutinin indicates the presence of O-linked carbohydrates.

F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognized by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

Using spleen cells of mice infected or immunized respectively with cercariae or antigen preparations of Schistosoma mansoni, S. haematobium or S. japonicum monoclonal antibodies (mAbs) were produced against the schistosome gut-associated antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen). Fusions nearly exclusively produced either anti-CAA (n = 25) or anti-CCA mAbs (n = 55) with a strong isotype restriction (IgM, IgG1 and IgG3) against both antigens, the majority of anti-CAA mAbs being IgG1 and the majority of anti-CCA mAbs being IgM. The mAbs, which on the basis of their selection were reactive with multiple carbohydrate epitopes of CAA or CCA, were applied in different immunological techniques including immunofluorescence, a dot immunobinding assay and immuno-electrophoresis to study the epitope repertoire. Anti-CAA mAbs were found to be reactive with 5 different epitopes, none of which occurred as multiple epitopes on eggs. Anti-CCA mAbs, on the other hand, recognized at least 10 different epitopes, while 44% of anti-CCA mAbs recognized epitopes common to the adult worm and the egg. Both CAA-and CCA-epitopes were found to be developmentally expressed at the level of the tegument in cercariae, schistosomula and 5-day-old lung worms, but in the adult worm were primarily found in the gut. Thus, the production of panels of mAbs has not only resulted in the selection of reagents optimally performing in diagnostic immunoassays, but also allowed a more detailed study of the epitope repertoire of these important schistosome antigens.