In order to visualize low-pH compartments of Plasmodium berghei strains we have used a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) which concentrates in acidic compartments, and can be detected by immunocytochemistry with anti-dinitrophenol antibodies. We have demonstrated that in a P. berghei chloroquine-sensitive strain (N strain), DAMP accumulates in the endocytic vacuoles where haemoglobin degradation is occurring. These compartments which have recently been shown to concentrate 4-aminoquinoline drugs (Moreau, Prensier, Maalla & Fortier, 1986) have an acidic pH. Conversely DAMP was found scattered all over the cytoplasm in a P. berghei chloroquine-resistant strain; the same phenomenon was previously observed (Moreau et al. 1986) in the localization of a 4-aminoquinoline on this same strain. Monensin-induced swelling of acidic compartments (Boss & Morre, 1984) was used as a complementary method for the determination of low-pH compartments on P. berghei strains. All the data reported here suggest that chloroquine resistance in P. berghei RC may be related to an impairment in the acidification of endocytic vesicles.

Characteristics of four species of Eimeria isolated from Apodemus sylvaticus are described. From oocyst morphology, three are identified as E. apionodes, E. hungaryensis and E. uptoni, the fourth being unnamed. Cloning demonstrated that oocysts of E. hungaryensis were polymorphic. Previous literature relating to the taxonomy is discussed and discrepancies outlined. Species isolated from A. sylvaticus were not transmissible to Clethrionomys glareolus, but both E. hungaryensis and E. apionodes have been passaged through immunosuppressed laboratory mice, the former species more than 16 times. In both A. sylvaticus and immunosuppressed laboratory mice, endogenous development of E. hungaryensis occurred mainly in enterocytes near the tips of the villi in the first half of the small intestine, with a few parasites in the rest of the small intestine and into the large intestine. The pre-patent period was 2 days in both hosts but oocyst output was higher in the natural hosts. E. apionodes parasitized enterocytes on the sides or at the base of the villi, mainly in the last 90% of the small intestine with a few parasites in the large intestine. The pre-patent period was 7 days. Parasites, probably E. hungaryensis and E. apionodes, were also isolated from A. flavicollis.

Various factors that may influence routine and high levels of mosquito infection with cultured Plasmodium falciparum gametocytes are considered in this paper. One of the most important is the choice of an appropriate isolate, with facilities for cryopreservation and a good technique for initiation of cultures. The use of automated culture systems with strict adherence to detail and routine has eliminated much of the variability. The quality of the serum used for the culture of gametocytes and inclusion in the feed material for mosquitoes is of the highest importance. Blood collection for culture purposes must preferably involve alcohol as an antiseptic for cleaning donor skin or suitable receptacles. Mosquito blood meals should not include plasma with citrate phosphate dextrose or sera collected in microtainer tubes or from volunteers on proguanil-chloroquine prophylaxis. Sera of individuals on chloroquine alone do not influence transmission. Haemato-crits of from 5 to 10% permit the culture of equally infective gametocytes. It was impossible to predict the outcome of an infection in mosquitoes based on the number of female gametocytes or gametes. Within any experiment, the oocyst load initially increased, followed by a decline with progressively lower numbers of gametocytes accompanied by a progressive increase in the efficiency of transmission. Some of the variability of mosquito infection within an experiment was due to individual differences in the speed of blood digestion of the mosquitoes. A new membrane feeder is described with three different sizes to accommodate a variety of goals. The new design permitted more mosquitoes to feed within a limited period compared to the older more widely used feeder of equivalent capacity, and mosquitoes feeding on the new-type feeder were frequently better infected than those fed on the older type.

I wish to thank the Society for inviting me to give the Introductory Talk in this Symposium. It is going to centre around what might be called ‘epitope specific’ immunization and I want to draw your attention to a number of factors which will be bound to affect any new approach to the development of vaccines. To begin with, it is now clear that complex antigens, such as those associated with parasites, present many different epitopes to the immune system. These epitopes are ‘seen’ by different cell types using different receptors and epitope recognition can activate a number of different effector systems.

Metacyclic forms of Leishmania major and putative metacyclics of L. mexicana mexicana were found to occur in abundance in stationary phase cultures. These forms have been compared in several ways with promastigotes from mid-log phase cultures and, in the case of L. m. mexicana, amastigotes. Metacyclics are smaller, contain less protein and appear more active than other promastigotes. Both forms of promastigote respire at a high rate in the absence of exogenous substrate. The free amino-acid contents of the various forms of the two species have been analysed. They differ in detail but alanine was the major amino acid in all cases. The isoenzyme content of the different forms differed significantly. That of the putative metacyclics of L. m. mexicana was in several respects more similar to amastigotes than promastigotes, suggesting that the form is pre-adapted for life in a mammal. Metacyclics of L. major apparently did not divide in culture but transformed back over a period of 48 h to mid-log phase cells. The results provide further detail of the molecular differences between mid-log phase and metacyclic promastigotes and confirm that metacyclics are a distinct form in the life-cycle.

The modern biology era in which molecular analyses dominate and immunology, cell biology and molecular genetics are prominent, has created unprecedented opportunities for the vaccine developer. The need for new and improved vaccines against many infectious disease agents is also great, no more so than for the protozoan and helminth parasite scourges of the rural poor in the tropical, less-industrially developed world. Despite the opportunities and needs, no vaccine against any human parasite yet exists nor does any molecular vaccine against any parasite; this chapter is a general discussion on the reasons for this state of affairs that assuredly will change soon.

Over the past few years several experienced groups studying malaria have encountered significant problems with their particular rodent malaria–host system. This has involved, in some cases, periods during which the recovery of cryo-preserved parasite stocks and growth of bloodstream parasites was markedly inhibited and, in other cases, periods of drastically increased mortality rates. The common factor linking these incidents was that they coincided with alterations in the experimental animal diet used. in the experimental animal diet used. The inhibition of growth of cryopreserved stabilates or bloodstream parasites was abolished by supplementation with p-aminobenzoic acid (PABA) or by changing the diet used. Although the suppressive effects of diets lacking PABA on parasite growth have been known for over 30 years, the variation of PABA levels in modern laboratory animal feed concentrates is not well recognized. We have not established the exact cause of increased mortality, but it has been overcome by changing the diet used. We are documenting our experiences with this potential variable to forewarn workers in other laboratories of possible problems inherent in the use of different diets.

Host selection among humans by Anopheles punctulatus was studied in an area of Papua New Guinea endemic for malaria and filariasis. Blood films were made from the stomach contents of freshly engorged mosquitoes found resting on the walls of houses in which the parasite status of the occupants was known. Engorgement rates on humans were non-random but could not be consistently related to the parasite status of individuals in the houses for either malaria or filaria. In some households, anophelines preferentially fed on parasitaemic individuals while in other households aparasitaemic individuals were significantly more often selected. This finding is believed to reflect the fact that malaria and filarial infections in this endemic area are predominantly asymptomatic. There were no significant differences in axillary temperatures between malaria or microfilariae positive or negative individuals.

This paper demonstrates the use of a quantitative genetical technique to assess genetic variance in molluscicide resistance in a Millettia thonningii-Biomphalaria glabrata system. Results indicated that there were significant genetic variances in both snail size and survival time in 10 p.p.m. molluscicide suspension. The possibility of wider applications of this technique in examining the genetic basis of molluscicide or pesticide resistance in target species is discussed.

Sex ratio theory usually predicts an equilibrium sex ratio and equal proportions of males and females in a population, including the progenitors of the reproductive cells of protozoans. This proposal was tested with three species of malarial parasites of lizards, Plasmodium mexicanum of the western fence lizard, and P. agamae and P. giganteum of the African rainbow lizard, using single samples from naturally infected lizards, repeated samples from free-ranging lizards (P. mexicanum only), and repeated samples from laboratory maintained animals. Macrogametocytes were usually more abundant than microgametocytes, and were slightly larger, revealing a typically greater investment of resources by the progenitors of female reproductive cells. However, the proportion of microgametocytes varied among the three species and among infections within each species of Plasmodium. The sex ratio of gametocytes often remained constant within infections followed over time even if the absolute number of gametocytes was changing. However, the equilibrium sex ratio of gametocytes varied among those infections that had an unchanging microgametocyte proportion. Thus, although an equilibrium sex ratio apparently occurs for most infections, there appears to be no characteristic proportion of microgametocytes for any of the species. Potential explanations for this conflict with theory are presented.

The purpose of this work is to review the progress towards malaria vaccination that has been made over the last four or so years, and the prospects and difficulties as they now appear. Although some of the older literature will be referred to as necessary background, it is not treated here in any detail. The reader who wishes for a fuller historical perspective should see, for instance, Brown (1969), Cohen & Mitchell (1978), Desowitz & Miller (1980), Mitchell (1984), Miller, David & Hadley (1984), Heidrich (1986) and, specifically for a consideration of sporozoite vaccination, Nussenzweig & Nussenzweig (1984, 1986). Naturally, any summary of vaccination will lean on the immunology of the disease, but malaria immunology is not reviewed here in its own right. The reader requiring the most recent attempt to cover this field should see the work edited by Perlmann & Wigzell (1988); some individual chapters of that volume will be referred to below.

The characteristics of intra-lymphocytic Theileria isolated from African buffalo and from cattle that were infected with buffalo-derived parasites were evaluated using anti-schizont monoclonal antibodies (mAbs) and DNA probes. Antigenic differences were revealed by the reactivities of 27 mAbs with the buffalo-derived parasites isolated from different animals. Antigenic diversity was also seen with Theileria-infected lymphoblastoid cell isolates taken from the lymph nodes and blood of the same animals. Two DNA probes, selected from a genomic library of T. parva piroplasm DNA cloned in λgt11, showed specific hybridization to parasite DNA in Southern blots of restriction enzyme-digested, lymphoblastoid cells infected with buffalo-derived theilerial parasites. Genotypic differences between the buffalo-derived parasites were revealed by the restriction fragment length polymorphisms seen with hybridization of those probes to DNA from cloned and uncloned Theileria-infected cell lines. The evaluation of theilerial parasites derived from buffalo and from cattle which underwent typical T. p. lawrencei reactions, after being infected with buffalo-derived theilerial parasites, did not show any specific phenotypic or genotypic characteristics of these parasites that would distinguish them from T. p. parva and T. p. bovis parasites. The validity of these subspecies distinctions is discussed.

The biosynthesis of phospholipids is extensive in Plasmodium knowlesi-infected simian erythrocytes due to the synthesis of membranes by this single-cell eukaryote in a host erythrocyte devoid of any pathway for lipid biosynthesis. In the present paper, we show that the incorporation of [3H]glycerol, which reflects de novo biosynthesis, is better studied at 300 μM−1 mM than at the trace doses, since this non-physiological precursor does not modify the amount of phosphatidylcholine biosynthesis from [3H]choline. Time-course incorporation of radioactive glycerol, oleate, lysophosphatidylcholine, choline, and inositol in RPMI 1640 medium containing nutrients for lipid synthesis showed that the optimum incubation time for phospholipid studies is 60–90 min, after which radioactive incorporation slows considerably. On the other hand, studies with [14C]serine revealed that incubation for 2–3 h is necessary for isotopic labelling of phosphatidylcholine via phosphatidylserine decarboxylation and phosphatidylethanolamine N-methylation. Incorporation of the various fatty acids into individual lipids was related to the molecular species composing each of them. Studies with [14C palmitoyl] lysophosphatidylcholine showed a very fast intracellular release of radioactive fatty acids, which indicates a potent lysophospholipase activity. Taken together, these data define the indispensable conditions for an experimental system suitable for further studies.

Parasites with low host specificity are predicted to search for hosts in ways less specific than highly host-specific species. They may also be better dispersers. Unionicolan mites that parasitize mussels are positively phototactic, with some species becoming negatively phototactic when host substances are present. This response has been interpreted as an aid to host location and occurs in species where adult mites have a relatively permanent association with hosts. Three species of Unionicola (Unionicolidae: Acari) that differ in host specificity co-occur in freshwater mussels (Unionidae) in St Mark's River in north Florida.

The need for a vaccine(s) against cutaneous leishmaniasis and the populations at risk for whom such vaccines should be developed are briefly discussed. The current human vaccine studies are reviewed, as are some experimental mouse studies with emphasis on Leishmania major infection relevant to vaccine development. Based on the information available from the mouse model and those data which are being sought in human studies, the benign nature of the cutaneous disease, the ease with which L. major can be manipulated to yield the required material, and the ongoing practice of leishmanization which allows rapid evaluation of candidate vaccine(s), it is suggested that a vaccine, at least against L. major, is imminent in the not too distant future.

Populations of Bulinus globosus and Biomphalaria pfeifferi were studied in a river habitat in Zimbabwe over a period of 12 months. Data were obtained on the prevalences of infections of Schistosoma haematobium (also S. mattheei) and S. mansoni respectively. Population parameters showed the following patterns for both snail species. (1) A patchy distribution correlated with the distributions of aquatic plants. (2) Life-expectancies of only a few weeks. (3) Recruitment rates correlated with water temperature and showing a distinct seasonal peak. (4) Spatial variation in recruitment. (5) A redistribution of snails during the rainy season. Epidemiological parameters showed the following patterns. (1) Large seasonal variations in the prevalence of patent infections. (2) Evidence from size-prevalence curves that suggests a variable force-of-infection from man to snail, correlated with water temperature. (3) Prevalences of infection that were higher in the vicinity of (±60 m from) major water contact sites. Local prevalences of infection for B. globosus sometimes exceeded 50% and may have approached 100% if pre-patent infections are included. Snail numbers may limit transmission at these locations. Attention is drawn to the need to make field observations at an appropriate spatial scale and also to the implications for the effectiveness of focal snail control as a means of reducing transmission.

During 1987 a total of 1393 cattle was examined for Schistosoma nasale infection at the Kandy slaughterhouse, Sri Lanka. The overall prevalence was 12·6% (monthly range 3–17%). Based on the appearance of macroscopic lesions, 6 types (0–5) were recognized; type 5 being the most severe, with cauliflower-like growths obstructing the nasal cavity. Older bovines with 8 permanent incisors were more heavily infected (29·%) than younger ones with no permanent incisors (6·0%). The severity of the lesions increased also with the age of the animals. Observations on the localization of the lesions showed that the first sessile nodular areas appear on the medial septum, on the dorsal edge of the ventral nasal concha and on the lateral wall of the middle meatus of the nasal cavity. Later, they gradually spread over the whole mucosal surface of the anterior part of the cavity but were rarely found further than 10 cm posterior to the nasal opening. The histopathology showed that granuloma formation due to the presence of eggs was the most common feature of the respiratory mucosa.

Vaccination against human schistosomes in laboratory hosts is now a reality. A number of different parasite molecules have been shown to confer partial protective immunity against challenge infection with Schistosoma mansoni or Schistosoma japonicum in rodent or primate hosts. These antigens are unusually diverse in their structure and stage specificity. Interestingly, although all of the vaccine molecules characterized are situated in the tegument, their exposure on the parasite surface, in most instances, is transient and/or non-essential. The properties of four of these immunogens, glutathione-S-transferase (P26,28), paramyosin (Sm97), GP38, and GP18 are discussed. Despite the identification and recombinant synthesis of several promising protective antigens, vaccination of humans against schistosomiasis remains in the realm of fantasy. At the technical level, a major problem is the failure of any of the current vaccine immunogens and immunization protocols to induce levels of resistance sufficient for significant reduction of human infection or disease. Once this important hurdle is passed, human immunization trials should be attempted as the potential beneficial impact of a vaccine against schistosomiasis remains enormous.

Circulating haemocytes from Echinostoma paraensei-infected M line Biomphalaria glabrata snails, or from age- and sizematched control snails, were studied on plastic slides with phase-contrast optics. Granulocytes, hyalinocytes, and round cells were consistently present; granulocytes were further categorized as ‘fully spread' (FS) or ‘partially spread' (PS). Among control snails, the relative percentage and estimated number/mm3 of round cells declined significantly with increased snail size, and the corresponding values for both categories of granulocytes increased. At 1 day post-infection (p.i.) with E. paraensei, overall composition of the haemocyte population was relatively unaffected, but by 8 days p.i. infected snails had significantly higher relative percentages of round cells and PS granulocytes than controls. Because a marked increase in the number of circulating haemocytes is also evident by 8 days p.i., infected snails had approximately 12 times more round cells and PS granulocytes/mm3 of haemolymph than did controls. At 30 days p.i. the relative and absolute abundance of PS granulocytes was still significantly elevated, but otherwise haemocyte populations did not differ from control snails. Alterations in granulocyte size in infected snails were also noted. Infection with E. paraensei has a striking impact of circulating haemocyte populations and also increases the relative concentration of haemocytes with less ability to adhere to a foreign surface.

A reproducible and standardized assay for measuring the cytoadherence of knobby Plasmodium falciparum-infected red cells to amelanotic melanoma cells was developed. Adhesion was dependent on temperature, haematocrit, and parasitaemia. Addition of EDTA to the binding medium reduced adhesion. Removal of protease-sensitive molecules on the surface of the infected cell abolished cytoadherence, whereas removal of carbohydrate residues by treatment of cells with neuraminidase or galactosidase promoted adhesion. Calcium, magnesium, fibrinogen or fibronectin in the medium had no effect on adhesion nor was there any enhancement of adhesion by pre-loading infected cells with calcium. Serum was essential for good adhesion. Adhesion was species specific for target cells; human endothelial or amelanotic melanoma cells were suitable target cells whereas bovine cells were not. The amelanotic melanoma cell could be formalin-fixed and still retain its adhesion properties. The binding properties of formalin-fixed amelanotic melanoma cells were not identical to those of endothelial or unfixed target cells.