Sporozoites of Eimeria tenella were disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.

An in vitro culture of Plasmodium falciparum (isolate FCUP-1/RSA) was arrested in the trophozoite stage with α-difluoromethylornithine (DFMO), followed by sorbitol treatment to liberate intracellular parasites from infected erythrocytes. Most of the unlysed erythrocytes and ghost membranes were removed by filtration, after agglutination with anti-erythrocyte monoclonal antibodies or wheat-germ agglutinin. A highly purified parasite preparation was obtained after chromatography on an immuno-affinity column consisting of polystyrene particles to which anti-erythrocyte monoclonal antibodies had been adsorbed. Isolated parasites were free of surrounding erythrocyte membranes and structurally intact as assessed by transmission electron microscopy, SDS–PAGE and ELISA.

This paper generalizes the two-dimensional approximation of models of macroparasites on homogeneous populations developed by Anderson & May (1978), focusing on how the dispersion (the variance to mean ratio) of the equilibrium distribution of parasites on hosts is related to the stability of the equilibrium. We show in the approximate system that the equilibrium is stabilized not by aggregation, but by dispersion which increases as a function of the mean. Computer simulations indicate, however, that this analysis fails to capture properly the dynamics of the full system, raising the question of whether any two-dimensional system could produce an adequate approximation. We discuss the relevance of our results to several empirical studies which have examined the relation of dispersion to the mean.

The frequency of recombination between unlike genotypes is central to understanding the generation of genetic diversity in natural populations of malaria. Here we suggest a way of investigating the problem which could complement conventional biochemical approaches to the population genetics of malaria. Sex allocation theory is one of the most successful areas of evolutionary biology. A well-supported prediction is that progressively less female-biased sex ratios are favoured with more outcrossing; equal numbers of males and females being evolutionarily stable in randomly mating outbred populations. We present a simple game theory model to support the idea that outcrossing rates in malaria will be correlated with the sex ratio of gametocytes in the peripheral blood of vertebrate hosts. Blood films from epidemiological surveys and culture-adapted isolates from Madang Province, Papua New Guinea, were used to estimate average gametocyte sex ratio of Plasmodium falciparum in the area. The geometric mean proportion of males in the population was 0.18 (95% confidence limits: 0.15–0.22). From our model, we estimate that, on average, 36% of zygotes are the result of outcrossing. This estimate assumes that most microgametes released following exflagellation are capable of fertilization. If, on average, fewer than about 70% of microgametes are capable of fertilization (as is the case in at least one other species of Plasmodium), the observed sex ratio would be consistent with between zero and 36% of zygotes being the result of outcrossing. These estimates suggest that there is usually a numerically dominant genotype in the gametocyte population in a blood meal, and that a considerable amount of selfing is occurring in P. falciparum populations in the Madang region, even though it is an area of intense year-round transmission.

The relative fecundity of populations of Eimeria tenella was estimated by means of mixed infection using electrophoretic variation of glucose-phosphate isomerases (GPIs) as a genetic marker. A decoquinate-resistant strain with GPI-9 isozyme (DR-NIAH), a decoquinate-sensitive one with GPI-1 (DS-Iwate), and three decoquinate-resistant lines (No. 3, 4, and 5) derived from cross-fertilization between DR-NIAH and DS-Iwate, were used. The GPI phenotypes of the No. 3 and No. 4 lines are GPI-9, and that of No. 5 is GPI-1. Mixed infection experiments were performed between DR-NIAH and DS-Iwate, No. 3 and No. 5, and No. 4 and No. 5. DR-NIAH was predominant over DS-Iwate in the mixed infection, whereas, in single infections, the total oocyst output of DR-NIAH was similar to or less than that of DS-Iwate. Among three lines, No. 4 was predominant over No. 5, and No. 5 was predominant over No. 3 in the mixed infection. Relative fecundity between No. 3 and No. 5 and their patterns of oocyst output in single infections were similar to those in the mixed infection. In contrast, No. 5 produced more oocysts than No. 4 in single infections, suggesting that the oocyst production in the mixed infection may be influenced by the mutual interference or competition between the populations of E. tenella in the chicken caeca.

A structure called ‘veil’ in Theileria sergenti-, T. buffeli- or T. orientalis-infected bovine erythrocytes was purified from erythrocyte lysates by Percoll density-gradient centrifugation. On electron microscopical examination, the veils consisted of electron-dense material showing a periodicity of striations and were not surrounded by membranes. Analyses of veil proteins by one- and two-dimensional polyacrylamide gel electrophoresis revealed that the veils contain haemoglobin and several other basic proteins of molecular weight ranging from 15·5 to 46 kDa. By comparing the protein patterns of the veil with those of purified piroplasms and uninfected bovine erythrocytes, these basic proteins appeared to be of parasite origin. It would appear likely that the veils formed by intra-erythrocytic precipitation of haemoglobin and proteins excreted by the parasites. Differences in veil constituents were found between T. sergenti, T. buffeli and T. orientalis.

A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650–800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range. The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock. G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA. Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4. The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite. In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture. Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

I wish to thank the British Society for Parasitologv for the privilege of being your Chairman this morning and for assembling such a promising series of papers for this Autumn Symposium.

Two sets of oligonucleotide primers, one derived from a repetitive sequence and the other from the gene encoding a 67 kDa sporozoite antigen of Theileria parva, were used to amplify parasite DNA from the blood of T. parva-infected carrier cattle using the polymerase chain reaction (PCR). PCR amplification products were obtained from 15 carrier cattle infected with one of 4 different T. parva stocks. Successful amplifications were performed using DNA from 2 cattle infected with T. p. parva Pemba Mnarani, 10 cattle infected with T. p. parva Marikebuni, 2 cattle infected with T. p. bovis Boleni and 1 animal infected with T. p. lawrencei 7014. No amplification products were obtained from any of 7 cattle which had been infected with the T. p. parva Muguga stock. A synthetic oligonucleotide, which hybridized specifically to T. p. parva Marikebuni DNA among 6 T. parva stocks tested, was designed using sequence data from within the region of the T. parva genome amplified by the repetitive sequence primers. The oligonucleotide was used to probe PCR products and to increase the sensitivity and specificity of carrier animal detection. Southern blot analysis using a T. parva repetitive sequence probe demonstrated the existence of restriction fragment length polymorphisms between parasites isolated from T. p. parva Marikebuni-infected carrier cattle. The use of the PCR and other methods of carrier animal detection are discussed.

The incidence of congenital toxoplasmosis was determined by an ELISA in the litters of BALB/c mice which had been infected 8 weeks before mating, on day 12 of pregnancy, or on both these occasions. Of those mice given the infection for the first time on day 12 of pregnancy, 5 out of 6 gave birth to infected litters with approximately 50% of the individuals in each litter being infected. BALB/c mice which had been infected 8 weeks before mating did not give birth to infected litters, even if they were reinfected on day 12 of pregnancy. Following infection BALB/c mice were found to harbour significantly fewer tissue cysts than the congenic H-2 derivative BALB/K strain. However, chronically infected BALB/K mice also failed to produce infected litters, indicating that tissue cyst burden in the dam did not influence congenital infection at least on the BALB background. This study demonstrates that BALB/c dams chronically infected with Toxoplasma gondii, have immunity capable of protecting their embryos from congenital infection, even if the dams are reinfected during pregnancy. Our results demonstrate that the BALB/c mouse can be used as a model of human or ovine congenital T. gondii infection suitable for testing putative vaccines.

The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species of Eimeria which infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility to E. tenella and susceptibility to the other species: lines which produced most oocysts of E. tenella produced least oocysts of the other species and vice-versa.

A murine monoclonal antibody which reacts to 22 and 41 kDa Toxoplasma gondii surface antigens was employed in an IgM capture enzyme-linked immunosorbent assay (ELISA). A total of 125 patients' sera were tested in the monoclonal-based assay. When compared with a commercial ELISA test (Abbott Toxo-M EIA) which uses polyclonal anti-T. gondii antibodies, good correlation (Pearsons coefficient r = 0.91) was observed. The specificity of the assay was studied by testing a panel of control sera obtained from healthy individuals and blood transfusion donors; all sera gave negative results. Serum samples positive for T. gondii antibodies were treated with 2-mercaptoethanol (2-ME) to demonstrate the specificity of the test for IgM antibodies. Reactivity of these sera was lost after the treatment. The test is not subject to interference by rheumatoid factor as sera positive for rheumatoid factor were negative in the assay. Reproducibility was good with the coefficients of variation for within-day tests below 10% and not exceeding 18% for day-to-day tests. The monoclonal-based assay is simple to perform and appears to be a viable test for diagnosis of T. gondii infection.

Until recently, almost all studies of leishmaniasis epidemiology were qualitative and descriptive. But now that the natural history of many Leishmania parasites is quite well known, there is growing interest in quantitative analysis. In this paper I use mathematical models in conjunction with field data to try to answer a wider range of questions than has previously been possible with descriptive techniques, and to sharpen some of the outstanding questions for laboratory workers. This is done with reference to the persistence and resilience of canine leishmaniasis, the maintenance of virulence poly morphisms in Leishmania populations, and the possible existence of cycles of human kala-azar. I conclude by posing a set of problems under three headings: diagnosis of infection (as distinct from disease), natural immunity to Leishmania infection in the vertebrate host, and genetic variation in the parasite population. Some solutions from the laboratory can be found in the companion paper by Black (1992).

The influence of adult female body size of Anopheles gambiae s.l. on development of midgut and salivary gland infections by the parasite Plasmodium falciparum was investigated in a field study carried out in Tanzania. The proportion of mosquitoes infected during a blood meal was independent of size. However, the number of oocysts harboured by infected mosquitoes increased with size of the mosquito. The proportion of mosquitoes with sporozoites, and thus potentially infective to humans, was highest in intermediate-sized mosquitoes, whereas the largest and smallest mosquitoes were less likely to have sporozoites. This pattern is interpreted as a combination of high survival rate of large, uninfected mosquitoes and of low survival rate of mosquitoes infected with many oocysts.

Application of quantitative methods to the study of leishmaniasis epidemiology has allowed Dye (1992) to pinpoint important biological parameters which, if they could be accurately measured in the field, would contribute most to our knowledge of the spread of disease and key targets for control. Three areas in which laboratory-based research could impact most on leishmaniasis epidemiology were highlighted by Dye (1992): (i) the development of accurate diagnostic tools which can distinguish between current and past infection; (ii) to determine the underlying molecular/genetic basis to virulence polymorphisms in the parasite and study these in the context of field epidemiological studies; and (iii) to provide the molecular tools to measure genetic variation in resistance to infection in humans and in reservoir hosts of disease. This paper describes current progress in attaining these goals, highlighting first the work on isolation and field application of genomic and kDNA probes for species-specific diagnosis, and the development of PCR-based assays which can be performed under field conditions. At a more preliminary stage, studies are described in which variability in the major molecular determinants of virulence (lipophosphoglycan, GP63, and members of the HSP7O family of stress proteins) identified through studies of laboratory models of infection, is being measured in primary field isolates of Leishmania peruviana. To complete the picture, current progress in identifying and cloning the genes which control host resistance to leishmanial infection is described, along with field studies of multicase families of human disease in which linkage analysis using marker genes from the chromosomal regions bearing these genes can be used to find evidence for their role in determining disease phenotypes in man. The leishmaniasis epidemiology will be all down to the DNA. projected view from these studies is that the future of leishmaniasis epidemiology will be all down to the DNA.

Recombinant ribosomal DNA sequences were amplified by PCR and used as probes to perform a fingerprint analysis of total DNA from different Entamoeba histolytica isolates. RFLPs obtained with one of the probes, R-1, support previous proposals that pathogenic and non-pathogenic E. histolytica are closely related, yet genotypically distinct. Another probe, R-2, while not distinguishing between the two forms of E. hystolytica, was able to differentiate between them and E. moshkovskii, which has morphologically identical cysts and trophozoites. A third probe, BR-1, identified strain-specific RFLPs.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5–72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later–successive–stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I – most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from ‘super-susceptible’ cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf–). In these cats the peak mf levels were seldom above 10000 mf/ml. Group II – these cats had less than 10 000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III – these cats became mf – during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics. Group IV – in these cats there was a sudden decline in the number of mf, usually in the first year and after they have received a relatively small number of re-infections. Circulating adult B. pahangi antigen disappeared from the serum within a short time of the loss of mf and at autopsy no adult female worms were found. Group V – these cats do not become mf+ when infected. These groups are compared to the parasitological groups seen in areas endemic for infection with Wuchereria bancrofti.