Serum-free media were used to culture Plasmodium falciparum. A commercial preparation, Daigo's GF21 developed as a growth-promoting factor for many kinds of mammalian cells, and consisting of the 55–70% ammonium sulphate fraction of adult bovine serum, insulin, transferrin, ethanolamine and sodium selenite, was found to sustain growth of the parasite when Daigo's T was employed as a basal medium. The optimal Daigo's GF21 concentration for parasite growth was between 5 and 20% (v/v), with the best results at 10%. Differential counts indicated that Daigo's GF21 is essential for schizogony. Established serum-free medium, GIT, consisting of Daigo's T basal medium and Daigo's GF21, yielded good parasite growth without any supplementation. Growth-promoting factor derived from adult bovine serum in Daigo's GF21 was shown to be crucial to parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements for the parasite.

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.

The genetic structure of a population of the malaria parasite Plasmodium falciparum has been examined in a village in Tanzania. Seventeen alleles of the merozoite surface protein MSP-1 and 23 of MSP-2 were detected by the polymerase chain reaction (PCR) among the blood parasites of the inhabitants. Most infections contained mixtures of genetically distinct parasite clones. PCR was then used to examine individual P. falciparum oocysts, the products of fertilization events, in wild-caught mosquitoes. Forty-five out of 71 oocysts were heterozygous for one or both genes, showing that crossing between clones was taking place frequently, following uptake of mixtures of gametocytes by the mosquitoes. The frequency of heterozygous forms showed that random mating events probably occurred within mosquito bloodmeals between gametes belonging to different parasite clones.

Following cloning of Leishmania (L.) amazonensis kinetoplast DNA two recombinant clones were identified: one specific for L. (L.) amazonensis and the other specific for L. (L.) amazonensis and closely related isolates. DNA sequences from these clones were compared with those of other kinetoplastids and oligonucleotide primers were designed to be used in the polymerase chain reaction. A pair of these primers has been shown not only to be highly specific for L. mexicana complex isolates but can also be used to distinguish between L. (L.) mexicana and L. (L.) amazonensis isolates. These primers have been tested with water-lysed cultures, crude DNA extracts from human patients, potential host reservoirs, sandfly vectors and with cell pellets after isoenzyme characterization. The results of these tests indicate that the primers can be used specifically in the presence of excess host DNA originating from the majority of South American countries.

As part of a survey of human leishmaniasis in Nicaragua we examined phenotypic and genotypic characteristics of 40 Leishmania isolates. We identified 3 distinct parasites associated with cutaneous disease in this area; Leishmania panamensis (40% of cases), Leishmania braziliensis (33%), and a strain which exhibits the heterozygous isoenzyme and DNA fingerprinting patterns expected of a L. panamensis/L. braziliensis hybrid (27%). There was complete correlation between the isoenzyme and DNA data for each of the putative hybrids examined. All of the ‘hybrids’ were obtained from foci in the northern region of the country where L. panamensis and L. braziliensis occur sympatrically. These observations provide strong evidence for sexual reproduction in New World Leishmania populations and suggest that it is of taxonomic and epidemiological significance.

Transmission of Schistosoma mansoni was monitored by routine snail sampling for Biomphalaria pfeifferi and by supplementary cercariometric measurements in 4 neighbouring study areas in Machakos District, Kenya. After 1 year, extensive, population-based chemotherapy with a single dose of praziquantel was given in 3 areas, but only minimal treatment in the fourth. In the year preceding treatment, seasonal transmission of S. mansoni and other non-human trematodes occurred in all 4 areas, despite some ecological differences and the effects of earlier treatment campaigns in 1 of the study areas. After treatment of all infected subjects in one area in which there had been earlier chemotherapy campaigns, S. mansoni transmission remained very low. It was reduced for at least 2 years after chemotherapy targeted at either all heavily infected subjects or all infected school children, but it was unaffected in an area where treatment was restricted to those few very heavily infected cases at risk of developing, disease. Nowhere was transmission entirely eliminated by chemotherapy and that of non-human trematodes continued unabated. The snail data correspond well with the human, parasitological data. Targeting school children was as effective as more extensive campaigns, but chemotherapy alone never stopped S. mansoni transmission: reinfection was inevitable, at rates determined by ecological factors affecting snail populations.

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10−9 and 10−7 M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10−8–10−6 M and 10−7–10−5 M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 µM FMR-DFamide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0·5 µM EGTA was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high K+-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

The tegument of the human parasite Schistosoma mansoni is critical for parasite survival within the mammalian host. The role of protein kinase C (PKC), a major effector molecule in the phosphoinositide pathway, in maintaining the structural organization of this syncytial layer was examined in adult worms. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDB), phorbol esters that activate PKC, induced formation of surface vesicles as determined by light and scanning electron microscopy. Similar results were seen with sn-2-dioctanoyl-glycerol, a synthetic analogue of diacylglycerol. No effect was seen in parasites incubated with 4-a-phorbol ester or a isomers of PMA or PDB, compounds that do not activate PKC. Vesicle formation was reversible in parasites treated with sn-2-dioctanoyl-glycerol but not with phorbol esters. The tegument of male worms was more sensitive to the effect of phorbol esters than females. Transmission electron microscopy revealed vacuolization of the tegument. These data suggest that signal transduction pathways may have a critical role in the maintenance of the structural integrity of the tegument of parasitic helminths.

By using fluorescent probes the distribution and fluid properties of lipid and saccharide-bound molecules was assessed in the tegument of praziquantel (−) treated Schistosoma mansoni adult male worms. Our results show that higher amounts of glycoproteins and/or glycolipids are exposed in damaged areas of the membrane compared with undamaged ones. At damaged regions these molecules present high lateral diffusion coefficient and mobile fraction values which suggests that after praziquantel(−) treatment they are being shed by the worm into the medium. The lateral diffusion coefficient of the lipid analogue 5'-octadecanoyl aminofluorescein is unchanged in damaged or undamaged areas but the mobile fraction is significantly reduced at damaged areas. The immunological significance of these altered surface properties is discussed.

Macrophage-like defence cells (haemocytes) of the pond snail Lymnaea stagnalis mediate cytotoxicity through reactive oxygen intermediates (ROIs). This activity is NADPH-oxidase dependent, as in mammalian phagocytes during the respiratory burst. In this study, mother sporocysts of schistosomes, the compatible Trichobilharzia ocellata and the incompatible Schistosoma mansoni evoke in vitro ROI activities (detected by luminol dependent chemiluminescence, LDCL) from L. stagnalis haemocytes. S. mansoni is encapsulated by haemocytes and eliminated, whereas T. ocellata escapes encapsulation and survives. Both schistosomes were equally susceptible to in vitro oxidative damage from exposure to hydrogen peroxide and to ROIs generated by a xanthine/xanthine oxidase system. Protocatechuic acid, a specific antagonist of NADPH-oxidase, delayed the killing of T. ocellata and S. mansoni sporocysts by haemocytes of resistant snails (Biomphalaria glabrata and L. stagnalis, respectively). We conclude that ROIs take part in haemocyte-mediated cytotoxicity. However, neither a snail's capability to generate ROIs, nor a schistosome's susceptibility to ROIs, determine snail/schistosome incompatibility. Snail/schistosome compatibility is rather determined by the parasite's ability modulate haemocyte behaviour such that effective encapsulation and the generation of lethal concentrations of ROIs are prevented.

Contrapsin, a serine protease inhibitor (serpin) present in mouse serum, was compared with that found in adult Schistosoma mansoni worm homogenates, which although immunologically identical to contrapsin in mouse serum, had a higher molecular weight in Western blotting. Immunolocalization studies demonstrated parasite-associated contrapsin on the surface and interstitial cells of adult male worms. After extraction of these parasites with Triton X-114, contrapsin was found in the aqueous phase of the detergent, suggesting it is unlikely to be an integral membrane protein. Treatment of adult worms with deoxycholate resulted in a change in the electrophoretic behaviour of worm-derived contrapsin. Parallel studies with trypsin suggested this was due to interaction of the serpin with a protease. Using porcine pancreatic trypsin as a model for a putative schistosome protease reacting with contrapsin, we have shown that trypsin, following complex formation with contrapsin, loses immunogenicity. Thus, when contrapsin–trypsin complexes were used as immunogen, the resulting antisera contained antibodies to contrapsin and contrapsin–trypsin complexes only, and none to native trypsin. Thus, epitopes characterizing native trypsin were presumably either masked following complex formation with contrapsin, or their processing and presentation to antigen presenting cells was suppressed, so that an antibody response was not mounted against them. These observations encourage speculation that S. mansoni may be elaborating an immune evasion strategy whereby immunologically sensitive proteases are first complexed with host serpins, which would render them immunogenically inert, and then cleared from the circulation by the host's reticulo-endothelial system. In this way the immune system would be unable to ‘see’ sensitive parasite proteases sufficiently to mount a response against the parasite.

We provide evidence from isoenzyme analysis, hybridization with repetitive DNA probes, behavioural studies and morphometrics that 4 trypanosome isolates from Glossina morsitans submorsitans in The Gambia constitute a new species now named Trypanosoma (Nannomonas) godfreyi. The bloodstream trypomastigotes of T. (N.) godfreyi are relatively small with a mean length of 13·7 μm (range: 9·1–21·8 μm) and a mean width of 1·65 μm (range: 0·65–2·69 μm). There is no free flagellum and the marginal kinetoplast is subterminal to a rounded posterior end; the undulating membrane is usually conspicuous. As with other Nannomonas, T. godfreyi developed in the midgut and proboscis of Glossina and infections matured in 21–28 days in laboratory G. m. morsitans. In The Gambia the normal vertebrate host appears to be the warthog, Phacochoerus aethiopicus, although elsewhere other wild and domestic suids may also be implicated in the life-cycle. T. godfreyi was identified unequivocally using a 380 bp DNA probe specific for a major genomic repeat sequence; its isoenzyme profile distinguished it clearly from T. simiae and three strain groups of T. congolense: savannah, riverine-forest and kilifi.

Strongyloides ratti has a complex life-cycle with two adult generations, one free-living and dioecious and one parasitic and female only. The parasitic females reproduce by parthenogenesis, but it is unclear whether this is mitotic or meiotic in nature. This question has been addressed genetically by analysing the progeny of parasitic females that were heterozygous at an actin locus for evidence of allelic segregation. Such progeny were similarly heterozygous showing that segregation had not occurred. It was therefore concluded that reproduction in the parasitic female of S. ratti is functionally mitotic.

To compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS–polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31·5–34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

Host immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs. Guinea-pig IgG was also found in saliva of the feeding ticks. The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay. Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35–42% of the antibody activity in guinea-pig immune serum remained in the tick samples. The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland. Such a mechanism could involve lgG binding proteins (1GBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG–agarose affinity column. In female ticks, the Mr of 1GBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa). The existence of 1GBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g. host immunoglobulin, from the body during feeding. This mechanism may be a part of the tick self-defence system.

A multi-subunit antigen (native Mr > 200 kDa, reduced Mr 97–100 kDa) has been identified in homogenates of Trichinella spiralis larvae using affinity-purified rabbit anti-keyhole limpet haemocyanin (KLH) antibodies and its cross-reactivity with KLH was confirmed by competition blotting. The antigen was not present at the larval surface but was exposed after treatment of the larvae with the detergent cetyltrimethyl ammonium bromide (CTAB) which removed the surface coat. This correlated with a significant decrease in insertion of the surface-restricted fluorescent lipid probe AF18, indicating that the surface coat must be lipidic in nature. Unlike KLH, the larval antigen blotted onto nitrocellulose was itself periodate insensitive. Periodate treatment of whole larvae, however, resulted in shedding of the surface, to which anti-KLH antibodies then bound intensely. Anti-KLH antibodies also recognized three (49, 55, 108 kDa) of the four most dominant antigens in excretory–secretory (ES) products of cultured larvae, whose excretion–secretion was increased with CTAB. The nature, location and function of the antigen is discussed.