The lack of correlation between parasitaemia and anaemia in severe malaria indicates that factors in addition to schizont rupture or erythrophagocytosis contribute to anaemia. We asked whether malaria toxin (MT) from Plasmodium berghei or P. chabaudi might impair erythropoiesis. Daily intraperitoneal injection of MT into C57BL/6 mice induced a transient reduction of RBC values by 25–30% after about 2 weeks, followed by increased haematopoiesis in the spleen as compared to mice receiving uninfected RBC preparations. There was a 3 (P. berghei) to 8-fold (P. chabaudi) increase of total proliferative activity in the spleen. Flow cytometric analyses showed that this was accompanied by some differentiation of TER-119 positive erythroid cells and of Gr-1 positive myeloid cells. Erythroid and myeloid progenitor cell-derived colony assays confirmed these results and revealed an increase in the number of CFU-E ([les ]200-fold), BFU-E ([les ]10-fold) and CFU-GM ([les ]20-fold) in the spleen of MT treated mice, as compared to controls.

Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39°C in the vertebrate host to 20°C) and a concomitant pH increase (from 7·3 in mouse blood to 8·0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise non-permissive pH of 7·3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogenesis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.

The combination of pyrimethamine and sulfadoxine (PSD or Fansidar) represents one of the most important chemo-therapeutic agents currently used to treat falciparum malaria. To investigate the molecular basis of resistance to PSD, reliable in vitro drug assays are required to permit correlation of resistance levels with different genotypes. We describe here protocols that permit accurate evaluation of IC50 values for sulfadoxine (SDX) inhibition of Plasmodium falciparum. Historically, tests for this drug have suffered from poor reproducibility and extreme variability in reported values. We have examined a series of variables, including serum-containing versus serum-free media, erythrocyte source, pre-test growth conditions, test components and post-test processing. We define conditions which better control the levels of the drug antagonists folate and p-aminobenzoate, yielding reproducible differences between lines of P. falciparum with differing alleles of the dihydropteroate synthetase gene, which encodes the target enzyme of SDX. We also use this assay to demonstrate a marked difference in the response of different parasite lines to antagonism of SDX inhibition by exogenous folate. The ability to measure reliable IC50 values for SDX inhibition should greatly facilitate further experiments to explore the molecular basis of Fansidar resistance.

The papers in this volume draw attention to both new and recent information on the mechanisms employed by infectious pathogens to underpin their survival in the immunocompetent host and to facilitate their transmission between hosts. Classical survival strategies include induction of immuno- suppression, antigenic variety and variation, host antigen sequestration, molecular mimicry, antibody destruction and invasion of cells or privileged sites. To these we can now add novel and diverse mechanisms with which the invader may manipulate the host for its own ends. They range from making use of a single molecular component of the immune system, through more sophisticated mechanisms of evasion to modulation of the immune response in the pathogen's favour, particularly by manipulation of T cell subsets and cytokine fluxes. There are then examples of pure exploitation of the adaptive immune system by the generation of specific humoral and cell-mediated responses that prolong the invader's survival and aid transmission. The order of the chapters in this volume is intended to reflect this increasing level of complexity.

It is our hope that the studies described in this multi-disciplinary assemblage of papers will stimulate further research in this important area. We would like to thank all our contributing authors and the anonymous refereees for their commitment and help in seeing this project through to completion.

We report the molecular characterization of a middle repetitive DNA sequence, named C6, isolated from the Trypanosoma cruzi genome. C6 appears to be a composite repeated element since 3 subregions may be defined within it on the basis of sequence similarities with other T. cruzi genomic sequences. Sequences homologous to C6 are interspersed in the genome and can be mapped out on most chromosomal bands of different T. cruzi strains. The copy number of the C6 element is about 1000 per haploid genome. Given the species specificity and different genomic distribution of C6 homologous sequences among the T. cruzi strains the C6 element could be a useful probe for diagnosis and typing of parasites. C6 is a polymorphic marker with potential as a tool for physical mapping of the T. cruzi genome.

The taxonomic situation in the genus Tritrichomonas is the subject of controversial discussion: potentially T. foetus and T. suis, the tritrichomonads from cattle and swine, respectively, could belong to the same species. In order to shed some light on this question, a molecular biological analysis was performed. The 5·8S rRNA gene and the flanking internal transcribed spacer regions (ITS1 and ITS2) of 12 different isolates of 3 Tritrichomonas species T. foetus, T. suis and T. mobilensis were enzymatically amplified by PCR and subcloned. Also, the corresponding regions of the trichomonads Trichomonas vaginalis, T. tenax, T. gallinae and Pentatrichomonas hominis were included in this study. Sequence analysis of cloned fragments was used to compare the parasite isolates. The genus Tritrichomonas exhibited an extremely high degree of homogeneity. All T. foetus and T. suis isolates had identical sequences, and only 1 substitution was found in the ITS2 region of T. mobilensis. In contrast, the genus Trichomonas shared more diversity. The results obtained in this study support a possible future revision of the taxonomic classification of tritrichomonads.

Biochemical characterization of 137 Leishmania braziliensis isolates from South and Central America, and from selected endemic foci in Bolivia, Brazil and Colombia, performed by isoenzymatic electrophoresis using 10 enzymatic systems, showed a high enzymatic polymorphism (44 zymodemes obtained) based on the variation of a small number of enzymes. Cladistic analysis showed close links between the zymodemes within the L. braziliensis s.s. cluster. The position of 2 Colombian zymodemes obtained (MON*204 and MON*205) justify the inclusion of L. peruviana within the L. braziliensiscluster.

We compared 19 stocks of Trypanosoma brucei rhodesiense collected in 1991 and 1994 from Tanzania with representative stocks from other foci of Rhodesian sleeping sickness in Zambia, Kenya and Uganda. Stocks were characterized by isoenzyme electrophoresis, restriction fragment length polymorphisms in variant surface glycoprotein genes and random amplification of polymorphic DNA; the banding patterns obtained were coded for numerical analysis. In addition, the Tanzanian stocks were compared by pulsed field gel electrophoresis. Overall the Tanzanian stocks formed a homogeneous group and the predominant genotype isolated in 1991 was still present in the 1994 sample, although at a reduced level. The Tanzanian stocks were distinct from representative stocks from other East African foci. This observation does not support the proposal that there are northern and southern strains of T. b. rhodesiense, but is consistent with the view that T. b. rhodesiense stocks form a mosaic of different genotypes varying from focus to focus in East Africa.

Parasites have established numerous strategies for evading host immunity. Such tactics promote persistence and chronicity – factors that favour completion of the life cycle and transmission to vectors or the environment. This volume explores many facets of the interaction between host and parasite that will be critical to the development of meaningful vaccines capable of interrupting what has been accomplished over centuries of co-evolution.

In this paper the Leishmania (Viannia) braziliensis complex is defined as containing all species of the actual subgenus Viannia. Organisms of the L. (V) braziliensis complex are the causative agents of localized human cutaneous and mucocutaneous leishmaniasis in South America, much of Central America and some areas of North America. In our search for better species and subspecies diagnostic probes we focused our research on repetitive DNA, since it provides a greater number of target sites for hybridization. In this work we report the isolation and sequencing of a 1·8 kb DNA region, LbJ38, which is probably tandemly repeated or dispersed at least 4 times along one chromosome and is naturally present in L. (V) braziliensis genomic DNA. This region contains microsatellites and simple repeat DNA sequences and was isolated by screening a genomic DNA cosmid library with complex- and species-specific probes. No homology was found with other Leishmania microsatellite or repetitive DNA. The utility of this repetitive sequence and primers derived from it in the identification of L. (V) braziliensis is demonstrated. As far as we are aware, this is the first report of sequence characterized repetitive microsatellite and GC rich simple repeat DNA from the nuclear genome of New World Leishmania.

The ability of deproteinated malaria exoantigens from Plasmodium falciparum (Pf-MT) and P. berghei ANKA (PbA-MT) to activate murine haematopoietic cells was analysed in vitro. Malaria toxins (MT) of both plasmodium species induced cell proliferation and the production of IFN-γ in overnight and long-term (5 days) spleen and bone marrow cultures and a reduction of the number of TNF-α spot forming cells (SFC). When added to cells of malaria-experienced animals, MT decreased the number of IL-4 SPC and increased the number of IL-5 SPC. However, the same proliferative and IFN-γ induction properties as in naive cells were observed. Simultaneous addition of IL-2 and PbA-MT to spleen cells inhibited the proliferation but increased the IFN-γ production usually induced by IL-2. Flow cytometric analysis revealed that the addition of MT triggered an expansion of CD3+ and GR1+ cell populations. Our results suggest that malaria toxins of different species can induce an immediate and strong proliferation and a TH1-type cytokine release by murine cells, independently of previous in vivo priming.

Multiplication of virulent and avirulent Leishmania donovani promastigotes was inhibited by brain heart infusion broth (BHI) (3·7%) supplemented with haemolysed or whole blood (2·5–10%) or heat-inactivated foetal bovine serum (HIFBS) (10–20%). Peptone, glutamine, tryptophan, purine bases, ribonucleosides, deoxyribonucleosides, B-vitamins and vitamin C were evaluated for the reversal of this growth inhibition. Supplementation of BHI with folic acid further supplemented with haemolysed rabbit blood or HIFBS and haemin supported propagation in serial subpassages of 2 primary isolates and 3 other virulent strains of L. donovani and 1 strain of L. major. Folic acid supplemented BHI with 3% rabbit blood in solid phase with 0·7% agar also supported the colony formation of L. donovani. This manuscript documents the development of high efficiency media for the cultivation of L. donovani promastigotes in liquid phase and as colonies on agar plates using BHI as a basal medium.

Interactions between parasite genotypes sharing a host are poorly understood, but have important consequences for the epidemiology and evolution of the parasite. In mixed-genotype malaria infections, patterns of asexual replication and transmission favoured by natural selection may be different from those in single-genotype infections. The infectivity to mosquitoes of mixed-genotype and single-genotype infections were compared using 2 clones of Plasmodium chabaudi inoculated into mice either together or alone. Mice given mixed-clone infections received the sum of the inocula given to the single-clone controls. Mosquitoes were fed on the mice and the numbers of oocysts which developed were counted to assess transmission intensity. For 3 combinations of starting inocula and feed days, mixed-clone infections produced more oocysts per mosquito than the sum of the 2 single-clone infections. This effect was correlated with an increase in gametocyte density, but was less clearly related to asexual infection parameters. The results show that interactions between clones in mixed-clone infections can profoundly affect transmission.

To study congenital transmission of Toxoplasma gondii during acute and chronic infections, 4 pregnant Sprague-Dawley rats were each fed 10000 oocysts of the VEG strain. Toxoplasma gondii was recovered from 33, 55, 83 and 57% of rats (F1) when dams were inoculated at 6, 9, 12 or 15 days of gestation, respectively. Progeny of 15 congenitally infected female rats were examined for T. gondii. Toxoplasma gondii was recovered from tissues of 1 of 155 rats (F2) born to congenitally infected dams. A total of 4 (F2) females were mated; 0 of 40 (F3) rats born to them were infected. None of the acutely infected 4 dams that had given birth to congenitally infected litters produced congenitally infected offspring during the second pregnancy. Thus, unlike mice, evidence for repeated congenital transmission of T. gondii in the rat was found in <1% of cases. Of the 16 congenitally T. gondii infected pups with demonstrable tissue cysts, 5 were seronegative (<1[ratio ]4) in the Sabin-Feldman dye test and 5 were seronegative (<1[ratio ]20) in the modified agglutination test by the use of whole formalinized tachyzoites and mercaptoethanol.

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-dl-phenylalanine β-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and evidence obtained after partial purification, SDS–PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody ‘responder’ mice were significantly (P<0·001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

The persistence of Toxoplasma gondii tissue cysts in organs of cats (definitive host) and rodents (intermediate hosts) was studied. Nine cats, 12 rats, and 12 mice were fed T. gondii oocysts and their organs were digested in pepsin and then bioassayed for bradyzoites in mice. Of 9 cats killed 37 or 51 days after feeding 102 (2 cats), 103 (3 cats) or 104 (4 cats) oocysts of the VEG strain, tissue cysts were found in each cat; in the tongue of 9, in the heart of 5, in the brain of 4, and in the eyes of 1 cat. The dose had no effect on the distribution of tissue cysts in cats. Twelve rats were each fed 105 oocysts of the VEG strain of T. gondii and killed 21, 29, 64 or 237 days later. At each time-period, 11 tissues of 3 rats were pooled and bioassayed in mice. Tissue cysts were found in the brain, skeletal muscle, heart and kidneys of rats at each killing time; in the lungs, intestines, and mesenteric lymph nodes in 3 of 4 instances; in the tongue, liver, and eyes in 2 instances and in the spleen in 1 instance. Also, using the same procedures and sampling the same 11 tissues as used for rats, tissue cysts were seen in all organs except in the tongue and liver of 3 mice killed on day 82 after feeding the VEG strain. In 9 mice (3 with each strain) fed oocysts of the ME-49, GT-1, or P89 T. gondii strain and killed 62–130 days later, tissue cysts were found consistently only in the brain. Thus, in rats and mice, most tissue cysts were found in the brain and rarely in the tongue. This was in marked contrast to the distribution of tissue cysts in cats.

The role of the peritrophic matrix (PM) in the development of Leishmania major infections in a natural vector, Phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked PM formation. Surprisingly, the absence of the PM was associated with the loss of midgut infections. The chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. Most parasites were killed in chitinase-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

A general method of investigating parasite population genetics is presented and used to investigate the evolution of drug resistance in Plasmodium. The most important biological factor is the nature of the control, presumably through host immunity, of the malarial infection. Two models are examined: a ‘generalized immunity’ (GI) model in which immunity regulates the overall level of infection, and a ‘specific immunity’ (SI) model in which each clone within the infection is regulated independently. These models are used to investigate 3 critical factors in the evolution of resistance: (i) the frequency of resistant alleles in the population prior to drug use, (ii) the dynamics of resistance following drug application and (iii) the magnitude of threshold frequencies below which resistance will not evolve. These analyses also identify the implicit assumptions made in several previous models, reconcile their differing conclusions and allow a more informed debate about the practical application of drugs.

Leishmania are intracellular protozoan parasites which reside primarily, if not exclusively, in host mononuclear phagocytes. Several studies have demonstrated that infectious promastigotes rapidly and efficiently fix complement when they encounter serum components during their transmission to the mammalian host. Activation of the complement system by a microorganism can have 3 distinct biological effects. First, fixation of the terminal complement components can result in complement-mediated lysis. Second, fixation of the 3rd component of complement can lead to opsonization of the organism for uptake by phagocytic cells. Finally, the elaboration of the complement anaphylotoxins, C3a and C5a, can lead to inflammation. In the present chapter, we discuss the interaction of leishmania promastigotes with the complement system. We show that infectious promastigotes avoid the lytic effects of complement and resist fixation of the terminal complement components. At the same time, however, these organisms depend on fixation of opsonic complement to invade host mononuclear phagocytes efficiently. We discuss the mechanisms which allow metacyclic leishmania promastigotes to exploit the opsonic properties of complement and the receptors on macrophages involved in leishmania recognition. The role of complement mediated inflammatory processes in the host response to leishmania infection is an area which requires additional study.

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.