Mixed infections with malarial (Plasmodium gallinaceum) and filarial (Brugia pahangi) parasites were carried out in 8 trials with filaria susceptible (REFM) and filaria refractory (REP-RR) Aedes aegypti strains. A secondary infection with B. pahangi microfilariae (mff) by intrathoracic inoculation, reduced the development rate of a pre-existing P. gallinaceum infection. The level of reduction ranged from 9·5 to 49% in REFM and from 50 to 90% in REP-RR. An immune response against oocysts was seen as melanization in mosquitoes with a double infection in the strain refractory to B. pahangi (REP-RR) and a reduction in oocyst size in both mosquito strains. Melanization was not observed in mosquitoes infected only with P. gallinaceum. This may indicate that activation of the prophenoloxidase (PPO) cascade in response to mff in the haemolymph can also be addressed against oocysts in the midgut. No significant difference in the number of filarial parasites recovered was observed when comparing groups with a single or double infection. Retardation in development of filaria larvae was observed in mosquitoes with double infection (REFM strain), together with melanization and a higher rate of abnormal development. Nutritional deficiency caused by superinfection might also be responsible for the delay in filarial development and reduced oocyst size.

Classical studies of non-immune individuals infected with Plasmodium falciparum reveal that the infection may be regulated for long periods at a relatively stable parasite density, despite the enormous growth potential of a parasite that continually replicates within host erythrocytes. This suggests that the parasite population may be controlled by density-dependent mechanisms, and in theory the most obvious of these is competition between parasites for host erythrocytes. Here we evaluate the role of this mechanism in the regulation of parasitaemia, by modelling the basic population interaction between parasites and erythrocytes in a form that allows all the essential parameters to be estimated from clinical data. Our results show that competition cannot account for the total regulation of P. falciparum, but when combined with immune mechanisms it may play a more important role than is generally supposed. Further analysis of the model indicates that in the long term, parasite replication at low parasite densities can contribute significantly to the high degree of anaemia observed in natural infection, a conclusion which is not obvious from simple clinical observation.

Trypanosoma cruzi specific sequences were amplified by the polymerase chain reaction from total blood of human chagasic patients and normal individuals. A 330 bp fragment originating from kinetoplast DNA was specifically detected in most chagasic individuals. We tested the sensitivity and specificity of this method in normal and affected individuals attending the Evandro Chagas Hospital, Rio de Janeiro. The results of these tests were compared with serological diagnosis performed using standard techniques, and in some cases with xenodiagnosis. We found that none of the serologically negative individuals gave any specific amplification product, whereas 55 out of 61 patients previously serodiagnosed as chagasic were positive using the PCR method (sensitivity: 90%). Xenodiagnosis, which is currently considered to be the most sensitive parasitological technique for Chagas' disease diagnosis, detected only 12 out of 28 serologically positive patients (sensitivity: 43%). The usefulness of the PCR method was further investigated with chagasic patients who had received anti-parasite treatment with benznidazole. It has always been difficult to evaluate the incidence of cure in such cases by serology, since a humoral response against T. cruzi antigens may remain for years even in the absence of the parasite. We observed a positive amplification result in only 9 out of 32 treated patients who remained reactive when tested using classical serology. These observations suggest that PCR is the most sensitive technique available for direct detection of T. cruzi in chagasic patients and that it can be a very useful instrument for the follow-up of patients after specific treatment.

In the present study ultrastructural immunocytochemical techniques have been used to localize the cells producing two soluble egg antigens present in the hatching fluid of Schistosoma mansoni, using two recently described monoclonal antibodies (MAbs). In the miracidium of S. mansoni, the one apical and the two lateral unicellular glands showed immunoreactivity for the egg antigens, using a post-embedding immunogold technique. Immunoreactivity was associated with the contents of vesicles for both the MAbs, although the intravesicular distribution was not identical. This difference in ultrastructural staining pattern provides additional evidence that the two MAbs are binding with different epitopes of the same antigen or with different antigens altogether. The finding of free unbound label around the embedded miracidium is consistent with secretion having been released from the glands.

Cytoadherence of Plasmodium falciparum-infected erythrocytes plays an important role in the pathogenesis of cerebral malaria. The identity of cell surface molecules on parasitized erythrocytes involved in cytoadherence is of great interest to understand the molecular basis of this mechanism. Peptide sequences derived from exofacial loops of the erythrocyte antigen band 3 from parasitized erythrocytes have previously been shown to inhibit cytoadherence. We now report that a non-repeated region of Pf155/RESA (residues 213–218) contains a hexapeptide motif being highly homologous to cytoadherence inhibitory sequences from band 3. Synthetic peptides containing the hexapeptide motif of Pf155/RESA inhibited the binding of P.falciparum-infected erythrocytes to melanoma cells in vitro. Furthermore, individuals residing in malaria-endemic areas have antibodies reactive with epitopes involving these motifs in band 3 and in Pf155/RESA.

The size of the infecting inoculum may influence the severity of malaria, but evidence is scarce. Malaria therapy records provide a unique source of information on induced malaria in people. The therapy was given to large numbers of neurosyphilis patients and the malaria was left untreated as long as possible. Data from patients treated at the Horton Hospital, Epsom 1923–60 with a single strain of vivax malaria were analysed to assess the influence of inoculum size on severity of disease. Malaria was induced by mosquito bite, blood inoculation or direct sporozoite inoculation. The different measures of inoculum size were inversely correlated with pre-patent period, as expected. Overall, information was available on a total of 563 non-immune patients who were not treated during the first 5 days of patent parasitaemia. No strong or consistent relationships were found between measures of inoculum size and any of the measures of severity used: neither parasitaemia levels, nor peak fevers, nor number of paroxysms of fever. In the largest data set, longer pre-patent periods were associated with tertian fever, spontaneous recovery and less use of modifying treatment. Difficulties in interpreting the results are discussed, particularly with respect to misclassification of both exposure and outcome variables. While an inoculum size-severity relationship cannot be ruled out, a strong relationship is very unlikely.

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultra-structurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radio-immunoassay of acid-ethanol extracts of the worms detected 8·3 pmol/g and 4·7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4·9 pmol/g and 4·3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

Small vessels showing inflammatory cell infiltrates were invariably observed in the vicinity of intact Toxoplasma tissue cysts within the brains of mice congenitally infected with the protozoan. Lymphocytes were observed in intimate contact with the luminal aspect of the endothelium, penetrating into the thickened basal lamina and in the perivascular area, which also contained macrophages and neutrophilic granulocytes. Rarely, lymphocytes were observed attached to the outer membrane of the host neurone which contained a Toxoplasma tissue cyst and within the inflammatory infiltrate associated with a disintegrating cyst. An hypothesis is presented which combines these morphological observations to explain the events associated with tissue cyst associated recrudescence of latent Toxoplasma infection in mouse brain.

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection.

Several years have elapsed since the publication by Johnson et al. (1989) of the cloning of a recombinant antigen from the cestode parasite Taenia ovis which stimulated high levels of protective immunity in sheep. A great deal of subsequent research and development was necessary to bring the fledgling vaccine to the point of being a registered commercial product. The results of these subsequent studies are dealt with briefly in this paper, including the results of field trials. The T. ovis vaccine was registered by the New Zealand Animal Remedies Board in February 1994. Where then is the commercial product ? This paper gives a background to market problems which have emerged through the politics (and realities) of the NZ T. ovis control campaign. It serves as notice that the best science dedicated to producing vaccines or products for parasitic, or other, diseases often faces significant hurdles in the real world of commerce and politics.

Progarnia archosauriae nov. gen., nov. sp. (Haemosporina: Garniidae) is described in the blood of the South American caiman, Caiman crocodilus crocodilus (Archosauria: Crocodilia). The parasite undergoes merogony and gametogony principally in leucocytes and thrombocytes, but also invades erythrocytes in which it produces no ‘malarial’ pigment. It thus shares features of Fallisia and Garnia which are, respectively, intra-leucocytic and intra-erythrocytic haemosporines of the family Garniidae in present-day lizards. This, and the antiquity of the order Crocodilia, suggests that it was from such a parasite that the existing reptilian and avian haemosporines evolved. An overall evolutionary pattern is suggested.

Adult parasites of Schistosoma mansoni reside within vertebrate mesenteric veins where they consume immense quantities of host glucose after transporting the sugar through their surface syncytium or tegument. Previously we obtained cDNA clones encoding two functional facilitated diffusion glucose transporter proteins expressed by S. mansoni adult worms (Skelly et al. 1994). Antibodies specific for one transporter (SGTP1) have been generated against an extrafacial and an internal domain of the protein and used to localize the protein by light and electron microscopy. By light microscopy both antibodies stain a linear structure approximately 1–5 μm from the surface of the tegument of adult male and female schistosomes. Electron microscopic examination of frozen thin sections show binding of the antibodies to membranes in the base of the tegument and not to the membranes covering the outer surface or their invaginations. Analysis of the gold distribution suggests that the extrafacial domain is disposed toward the interstitial space beneath the tegument and the internal domain faces the syncytial plasm. The localization suggests that SGTP1 may function to transport free glucose from within the tegument and into the interstitial fluids that bathe the internal organs of these parasites.

Isoenzyme typing was used to study a number of oocyst isolates of Cryptosporidium parvum from different geographical locations and of human or animal origin. All isolates showed identical enzyme motility when glucose phosphate isomerase (GPI; 23 isolates tested) or lactate dehydrogenases (LDH; 20 isolates tested) was assayed. However, two isoenzyme forms were observed with phosphoglucomutase (PGM; 9 animal isolates showed one form, while 8/9 human isolates showed a second form) and hexokinase (HK; 4 human isolates showed one form and 6 animal isolates showed a second form). Thus, PGM and HK each exhibit 2 isoenzymes corresponding to 2 parasite populations associated with separate hosts. The data from this study, plus supportive evidence obtained by different methods and by independent researchers, lend support to the hypothesis that separate cycles of transmission of C. parvum may exist within human and animal hosts.

This paper describes the development of the first commercial vaccine for toxoplasmosis. The vaccine comprises live tachyzoites of the S48 ‘incomplete’ strain of Toxoplasma gondii and is deployed to control toxoplasma abortion in sheep. A discussion of protective immune mechanisms and recent studies on host responses to the vaccine is also included.

The current taxonomy of parasites in the genus Sarcocystis is largely based on morphological characteristics as well as on host specificity and life-cycle structure. Recently, phylogenetic analyses of partial ribosomal RNA (rRNA) sequences provided support for paraphyly of Sarcocystis. We have tested the validity of this hypothesis by sequencing the complete 18S rRNA genes of Sarcocystis arieticanis, Sarcocystis gigantea and Sarcocystis tenella and comparing them with gene sequences derived from other taxa of the phylum Apicomplexa. The results obtained from this study do not reject the hypothesis of monophyly of Sarcocystis species, although the bootstrap data were inconclusive for some species.

A method for phenetic analysis of karyotype data has been developed for Leishmania populations. Measurement of size difference between chromosomes recognized by a given DNA probe in different isolates led to the formulation of a Chromosome Size Difference Index (CSDI). The method was applied to phenetic analysis of 4 sets of chromosomes – each set being recognized by a different probe – in 37 L. (Viannia) peruviana isolates sampled along a North–South transect through the Peruvian Andes and, in 11 L. (V.) braziliensis isolates from the Amazonian forest (Peru, Bolivia and Brazil). Karyotype variability was better accounted for by CSDI than by a method based on disjunctive encoding of karyotype data. CSDI evidenced the nature of relationships between L. braziliensis and L. peruviana and it provided a coherent picture of geographical and genomic differentiation among parasite populations. The latter did cluster according to their geographical origin. L. braziliensis was found karyotypically more homogeneous than L. peruviana. Within L. peruviana, Northern populations were closer to L. braziliensis than to Southern L. peruviana populations. The validity of karyotypic populations, or karyodemes, was sustained.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immuno-fluorescence antibody test monoclonal antibody (MAb) IB5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

In a population of copepods (Cyclops strenuus abyssorum), with a naturally high prevalence of infection with procercoids of Diphyllobothrium spp., no difference in body size was found between infected and uninfected hosts. However, a significant reduction in the reproductive capacity of infected females was observed, 87% of uninfected females having developed eggs in their gonads and sacs compared with 21% of infected females. The feeding rate of infected copepods was relatively high soon after infection occurred, but gradually decreased to less than half that of uninfected animals. Respiration rate was also lower in infected copepods. Infected copepods showed reduced motility and impaired escape responses, which is likely to make them more susceptible to predation.

A polymerase chain reaction (PCR) based on the detection of the Lmet2 repeat sequence specific to members of the Leishmania donovani–complex is described. To improve PCR specificity, a post-PCR hybridization step is often performed but this usually involves an entirely new procedure with additional manipulations, expense and time. We have simplified this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed automatically as part of the PCR programme. The hybrids are afterwards detected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-linked immunoassay (PCR–SHELA), is rapid, able to detect less than 5 cultured parasites and is specific for parasites of the Leishmania donovani–complex. We also describe the application of PCR–SHELA to the detection of amastigotes in various tissues of infected laboratory animals.