The mini-exon gene repeats from three different strains of Endotrypanum schaudinni have been amplified by the polymerase chain reaction (PCR). Sequence analysis of the cloned products shows the gene and intergenic region to be identical in two of the strains (LV86 and M6159); the intergenic region from the mini-exon gene of the third strain (LV59) is significantly different. The LV86 gene and an intergenic probe from the LV59 mini-exon gene do not cross-hybridize with the mini-exon gene from New World Leishmania species. These data provide the basis of a PCR assay to detect E. schaudinni and distinguish it from New World Leishmania species, and which should be applicable to epidemiological studies in insect vectors and mammalian reservoirs. The identification of two different mini-exon gene repeats in E. schaudinni isolates is indicative of further strain variation within this species.

The effects of purified monoclonal immunoglobulins from control, or transmission-blocking anti-Pbs21 antibodies, upon the infection of Anopheles stephensi by ookinetes of Plasmodium berghei are compared. Anti-Phs21 antibody reduced mean intensity and prevalence of infection by 94·7 and 58·7% respectively if added to the infectious bloodfeed at a concentration of 100 μg/ml. Fab fragments were of similar efficacy. No transmission enhancement was detected with declining antibody concentrations. Addition to subsequent (second) feeds reduced mean oocyst intensity but not prevalence. The reduction in blockade declined from 41% at day 2, to 4% at day 8. Second bloodfeeds, containing control globulin taken 4 or 6 days (but not 2 days) after infection, increased sporozoite burden in the salivary glands. At all times anti-Pbs21 reduced sporozoite number in the thorax compared to time-matched controls, but again highest gland intensities were obtained when the second bloodfeed was given on day 4. We conclude that second bloodfeeds containing transmission-blocking antibody simultaneously serve two opposing roles, (1) inhibition of parasite development and (2) the supply of nutrients which permit more sporozoites to be produced by each oocyst.

A new isolate of Trypanosoma musculi has been derived from organisms recovered from Mus spretus captured in Lisbon, Portugal. The time-course and profiles of infection with this isolate in inbred mice have been compared with those obtained with the existing Partinico II isolate. Infections with the Portuguese isolate are less intense, and controlled more quickly than those with the Partinico isolate. As with the latter, there are marked mouse strain-dependent influences on infection with the Portuguese isolate, but these strain-dependent characteristics differ considerably with each isolate. For example, NIH mice were the most susceptible to the Partinico II isolate, but virtually refractory to the Portuguese isolate. Mice exposed to infection with one isolate show complete immunity to both homologous and heterologous challenge infections. These striking interactions between host and parasite genotype are discussed in terms of immunological influences on infection.

Nucleosome filaments of two stages of the life-cycle of Trypanosoma brucei brucei, namely bloodstream forms and procyclic culture forms, were investigated by electron microscopy. Chromatin of bloodstream forms showed a salt-dependent condensation. The level of condensation was higher than that shown by chromatin from procyclic culture forms, but 30 nm fibres as formed in rat liver chromatin preparations were not found. Analysis of histones provided new evidence for the existence of H1-like proteins, which comigrated in the region of the core histones in SDS–PAGE and in front of the core histones in Triton acid urea gels. Differences were found between the H1-like proteins of the two trypanosome stages as well as between the core histones in their amount, number of bands and banding pattern. It can be concluded that T. b. brucei contains a full set of histones, including H1-like proteins, and that the poor condensation of its chromatin is not due to the absence of H1, but most probably due to histone–DNA interaction being weak. It is obvious that structural and functional differences of the chromatin exist not only between T. b. brucei and higher eukaryotes, but also between various stages of the life-cycle of the parasite. It is therefore not adequate to investigate the chromatin only of the procyclic culture forms as a model for all stages of the life-cycle of T. b. brucei.

A technique is described for the efficient feeding of Rhipicephalus appendiculatus nymphae on cattle blood through an artificial membrane bearing tactile and olfactory stimuli. The effect of four anticoagulation methods on the feeding of nymphae was compared and heparinized blood was found to be the most efficacious, followed by defibrinated blood. Blood treated with acid citrate dextrose (ACD) or ethylenediamine tetraacetate (EDTA) inhibited nymphal feeding. Nymphae fed on heparinized and defibrinated blood obtained engorgement weights within the range of ticks fed on mammalian hosts and they subsequently moulted and fed normally as adults and produced viable eggs. Nymphae fed on membranes using either defibrinated or heparinized blood infected with Theileria parva piroplasms developed salivary gland infections as adult ticks and transmitted East Coast fever (ECF) to susceptible cattle. There were indications that T. parva-infected defibrinated blood was not as infective to the feeding nymphae as the infected heparinized blood. When T. parva-infected heparinized blood was used to feed nymphae through membranes in two experiments, it was found that the infections in the resultant adult ticks could be comparable to those of nymphae fed on donor cattle, but were usually lower. The membrane feeding technique will enable the study of factors affecting the tick and T. parva transmission without the complication of host/T. parva interactions and could be useful for both tick maintenance and Theileria parasite isolation and maintenance.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ixodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 °C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.

The lung schistosomulum of Schistosoma mansoni is the target of protective immunity in mice singly vaccinated with irradiated cercariae. Since the effector responses are T cell-mediated, their initiation requires the release of antigens from the intact parasite. We have used the technique of biosynthetic labelling with ‘35S’methionine, before and after transformation of the cercariae, to analyse the kinetics of protein synthesis and release by the schistosomulum. In addition, the proteins present in the soluble fraction of the parasite and those released during in vitro culture have been characterized. During a 7-day culture period schistosomula derived from labelled cercariae lost proteins most rapidly within the first 3 h after transformation. Two proteins of molecular weight 61 and 20 kDa were dominant and may correspond to areas of proteolytic activity. Analysis of the rate of protein synthesis of schistosomula labelled after transformation revealed four different phases, which may relate to the developmental processes occurring in vivo. During the first 24 h, synthesis was very low, increasing to a plateau and then rising to a peak at day 8; thereafter the rate declined rapidly. Whilst some stage-specific synthesis of proteins was detected in the soluble fractions of the parasite bodies, the pattern of proteins released by cultured larvae was remarkably uniform. At least 15 proteins were detected by autoradiography with bands at 61, 45 and 20 kDa being particularly prominent. These proteins merit further study as potential mediators of the protective immune response.

Infection by Schistosoma mansoni resulted in morphological and biochemical changes to the foot of its intermediate host, Biomphalaria glabrata. Migration through, and emergence of cercariae from, the foot was observed and evidenced by lesions on the ciliated foot surface. This was accompanied by a significant decrease in the velocity of movement by infected individuals. In vivo31P NMR spectral analyses demonstrated that the foot of infected snails had a lower phosphoarginine (PA)/adenosine 5' triphosphate (ATP) ratio than that of uninfected controls. Moreover, kinetic experiments, employing saturation transfer, demonstrated the pseudo-first-order rate constant for the arginine kinase-catalysed exchange reaction in the forward direction, that is, PA→ATP was decreased by infection. The reverse reaction was not observed by the NMR methods used. PA was depleted upon exposure to hypoxic conditions suggesting its traditional role in preserving ATP level. Partly oxidized metabolic end-products were not observed in snails maintained under aerobic conditions, but succinate, propionate, acetate and lactate rapidly accumulated under hypoxic conditions.

An earlier paper (May, 1977) developed a theoretical framework for exploring the consequences of dioecy for the population dynamics of schistosomes, assuming an unbiased sex ratio. This paper extends the analysis to biased sex ratios, as have been reported in practice. We consider the relationships of the mean number and distribution of worms among hosts, the sex ratio, and the mating system (monogamous or polygamous) to: (i) the female mating probability, Φ the prevalence of mated female worms. Ω: and (iii) the mean number of mated female worms per host, ξ. Among other results, we show how high values of Φ are associated with male-biased sex ratios and polygamous mating; that Ω is independent of the mating system and is relatively unaffected by the sex ratio; and that ξ is maximal for unbiased sex ratios given monogamous mating, and for female-biased sex ratios if mating is polygamous. These results, together with the confounding effects of the mean number and distribution of worms, are described in detail in the main body of the paper.

The effect of the microtubule inhibitors coichicine (1×10−3 M) and tubulozole-C(1×10−6 M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of T1 secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type I cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, ‘yolk’ globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

Antigen preparations from three isolates of Trichinella spiralis (sensu-stricto) have been tested for their ability to immunize mice against homologous and heterologous challenge infections. Immunization was measured by accelerated expulsion of adult worms, reduction in female worm fecundity, stunting of growth, and level of antibody responses before and after challenge. Antigens of two isolates, from London (L) and Poland (P), immunized effectively against homologous challenge, adult worm recoveries being reduced by 64% and 51% respectively within 6 days. Antigen from a Spanish(S) isolate gave no homologous protection in terms of worm recovery at day 6 and only 43% reduction at day 8. L- and P-isolate antigens immunized well against heterologous challenge (day 6 worm recoveries reduced by 66% and 59%) but similar protection against S-isolate challenge was not seen until day 8. Immunization with S antigen gave better protection at day 6 against L-isolate challenge (77%) than against challenge with P-isolate (37%). These results imply considerable, but not complete, antigen cross-reactivity between the isolates and this was reflected in antibody responses. These variations in immunogenicity and immunization potential are discussed in terms of constraints on the use of vaccines against parasites that are widely distributed geographically.

In Guatemalan villages people commonly rear pigs, and both hosts may be infected with Ascaris. This study was designed to ask whether both humans and pigs are potential hosts in a single parasite transmission cycle in such villages, or alternatively, if there are two separate transmission cycles, one involving pigs and one involving human hosts. Parasites were collected from both host species from locations in the north and south of Guatemala. Allelic variation in the nuclear genome of Ascaris was measured using enzyme electrophoresis, while mitochondrial DNA (mtDNA) sequence variation was quantified using restriction mapping. Low levels of enzyme polymorphism were found in Ascaris, but allele frequencies at two loci, mannose phosphate isomerase and esterase, suggest that there is little gene exchange between parasite populations from humans and pigs. MtDNA haplotypes fall into two distinct clusters which differ in sequence by 3–4%; the two clusters broadly correspond to worms collected from humans and those collected from pigs. However, some parasites collected from humans have mtDNA characteristic of the ‘pig Ascaris’ haplotype cluster, while some parasites collected from pigs have mtDNA characteristic of the ’ haplotype cluster. These shared haplotypes are unlikely to represent contemporary cross-infection events. Patterns of phylogenetic similarity and geographical distribution of these haplotypes suggest, instead, that they are the result of two historical introgressions of mtDNA between the two host-associated Ascaris populations. The results clearly demonstrate that Ascaris from humans and pigs are involved in separate transmission cycles in Guatemala.

A recombinant Echinococcus multilocularis antigen (II/3–10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3–10 antibodies identified the corresponding epitopes on bands with identical Mr in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3–10 antibodies reacted with antigens of different Mr from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

The inheritance of response phenotype to Heligmosomoides polygyrus was investigated in Fl hybrid progeny of fast and slow responder mouse strains. The fast responses of SJL(H-2s) and SWR(H-2q) mice were mediated by dominant genes complementing each other in Fl hybrids which lost worms earlier and produced faster parasite-specific IgG1 antibody responses than either parent. However, the response of Fl hybrids from crosses of C57BL/10 (B10, H-2b) mice with either SJL or SWR differed from that of the parental strains and from each other: (B10 × SWR)F1 lost worms earlier than SWR whilst (B10 × SJL)F1 lost worms later than SJL mice. The Fl progeny of SJL mice with congenic strains B10.G (H-2q) and B10.S (H-2s) lost worms as quickly as SJL. Therefore, the response phenotype mediated by the genome of SJL mice was unaffected by H-2 heterozygosity (with H-2q) or homozygosity (H-2s) despite heterozygosity with B10 background genes, but was slowed significantly by heterozygosity with H-2b. All hybrids involving heterozygosity with B10, irrespective of MHC haplotype or background, failed to clear worms completely, in each case a proportion of mice harbouring residual worm burdens after loss of worms from parental strains.