The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4–6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

The C57BL6 strain of mice is highly susceptible to Plasmodium berghei sporozoite infections and consequently requires repeated immunizations with irradiated sporozoites to obtain protective immunity. After a live sporozoite challenge in the immunized hosts, hepatic-stage parasites found in the liver after 48 h are of different sizes – small schizonts corresponding to blocked forms (derived from irradiated sporozoites), and schizonts of intermediate size (derived from live sporozoites). Large schizonts corresponding to mature hepatic forms are found only in unimmunized but challenged C57BL6 mice. Using monoclonal and polyclonal antibodies directed to liver-stage parasites, different patterns of binding reactivity to the above forms are observed. More than 20% of the irradiated sporozoites transform into blocked forms after immunization and persist in the liver. Upon sporozoite challenge in such immunized animals the rate of transformation of sporozoites into hepatic parasites is less than 2%. These observations shed light on the fate of live sporozoite development in irradiated sporozoite-immunized C57BL6 mice.

Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.

We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed.

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

This study compared the biological and genetic properties of a bovine (NC-SweB1) and a canine (NC-Liverpool) isolate of Neospora caninum. A mouse model for CNS infection demonstrated marked differences in pathogenicity between the isolates. NC-Liverpool induced severe clinical signs of neosporosis in 57/58 mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss. In contrast NC-SweB1 induced similar but less severe symptoms in a much smaller proportion of mice over the same time-period. Statistically significant differences were observed between the isolates in the response (mean weight loss) of mice through time to the different doses inoculated. Histopathological effects on brain tissue reflected the isolate-based differences described above. NC-Liverpool infection resulted in intense inflammatory infiltrates and highly necrotic lesions whereas NC-SweB1 induced a milder meningoencephalitis. Passage in cell-culture over a period of 14 months did not affect the pathogenicity of NC-Liverpool. Immunoblots showed that antibodies to N. caninum appeared earlier in mice inoculated with NC-Liverpool than with NC-SweB1. Finally, RAPD–PCR analysis of NC-Liverpool DNA generated profiles distinct from that observed with DNA from NC-SweB1 or Toxoplasma gondii. In summary this study provides evidence for significant biological and genetic differences between 2 isolates of N. caninum.

To investigate the phenomenon that PCR of Leishmania (V.) lainsoni minicircles using primers B1 and B2 gives anomalous small-sized products, unlike all other members of the Leishmania Viannia subgenus, cloned kDNA minicircles from L. (Viannia) lainsoni were sequenced using fluorescent dye terminator reactions. The sequence of L. (V.) lainsoni where the primer B2 would be expected to bind, was different from the other members of the L. Viannia subgenus, matching in only 7 out of 19 bases with the sequence of L. (V.) braziliensis at this position. The sequence obtained from the cloned minicircles enabled the design of a new primer which, when combined with B1, allowed the amplification of full sized minicircles in L. (V.) lainsoni, but not other members of the L. Viannia subgenus. Comparison of the sequence obtained for Leishmania (V.) lainsoni with other Leishmania minicircle DNA confirms that Leishmania (V.) lainsoni is more similar to members of the L. Viannia subgenus than to other Leishmania, but is distinctly different.

Trypanosoma rangeli can infect humans and the same domestic and wild animals and triatomine vectors infected by T. cruzi in Central and South America. This overlapping distribution complicates the epidemiology of Chagas disease because of the cross-reactivity between T. rangeli and T. cruzi antigens. We have studied T. rangeli strains isolated from different geographical regions using the mini-exon gene as a genetic marker. Two pairs of oligonucleotides directed to this gene were designed in order to detect specifically T. rangeli DNA by PCR assays. This assay was highly sensitive, able to amplify the target sequence using the equivalent DNA content of a single parasite as template, and demonstrated no cross-reactivity with T. cruzi DNA. T. rangeli SC-58 strain, isolated in southern Brazil, showed a distinct electrophoretic pattern from the other T. rangeli strains tested. Low stringency single specific primer-PCR (LSSP) assays were able to detect sequence polymorphisms at the mini-exon gene among T. rangeli strains. Sequence comparisons of this gene revealed that the SC-58 strain was genetically distinct from strains isolated in Central America and northern South America. In addition to insertion/deletion events, the presence of microsatellite repeats in the non-transcribed region of the gene contribute to the intra-species variability.

Survival and infectivity characteristics are described for cercariae of the echinostome Echinoparyphium recurvatum at 10, 15, 20, 25 and 30°C. Cercarial survival was markedly age- and temperature-dependent, maximum survival time being reduced from 68 h at 10°C to 12 h at 30°C, 50% survival times falling from 47·9 h at 10°C to 8·4 h at 30°C. The mean instantaneous per capita death rate of cercariae increased exponentially with temperature. Cercarial infectivity was also temperature dependent, the main effect of temperature being upon the rate at which infectivity diminished with increasing age of cercariae. Infectivity increased from zero at 10°C to a maximum at 25°C after which it declined to a low level at 30°C. Overall transmission efficiency of cercariae at each experimental temperature was calculated as the ratio of the mean instantaneous per capita death rate (μ) to the mean instantaneous per capita rate of infection (β). Transmission efficiency of cercariae was found to be maximal at 20°C and at a relatively high level between 10 and 25°C. Transmission efficiency was zero at 10°C and at a very low level at 30°C. These results suggest that the cercariae of E. recurvatum show transmission optima at water temperatures likely to be encountered in natural habitats in Britain and Europe.

The isolation of 2 genomic clones has allowed us to further characterize a Schistosoma mansoni serine protease designated SmSP1. The deduced amino acid sequence (248aa) considered as a ‘light chain’ encoding the active site, presents significant homologies with mouse plasma kallikrein and human factor I light chain. The secondary structure of SmSP1 ‘light chain’ is correctly predicted and may be sufficient by itself to constitute an active enzyme. The biological function of SmSP1 is unknown, however, the homology with 2 serine proteases suggests that SmSP1 may play a role in the evasion of the host immune response. This is supported by the presence of the native protein corresponding to SmSP1 particularly in schistosomula released products (SRP) and in male dorsal spines. The expression of this enzyme is differentially regulated throughout the parasite life-cycle. However, infected animals with S. mansoni did not produce specific antibodies to recombinant SmSP1. The lack of such response could be advantageous to the parasite by protecting itself from host effector mechanisms.

Nematode surface coat (SC) proteins and excreted–secreted products (E–S) are likely to play important roles in the host–parasite interaction and considerable similarities can be found in SC proteins and E–S products from certain plant and animal parasitic nematodes. Monoclonal antibodies raised to E–S products of plant-parasitic nematodes were shown to cross-react with E–S products and the surface coats of the animal parasites Trichinella spiralis and Haemonchus contortus. Most of the antibodies recognized carbohydrate epitopes but the activity of 2 MAbs (IACR–CCNj.2a.15 and IACR-Misec.8D.3) which recognized proteic epitopes in these nematodes were further characterized. Antibody 2a.15 recognized the SC and oral exudate of Meloidogyne incognita, T. spiralis and H. contortus. This antigen was also immunolocalized in the lining of the oesophagus and gut and in the exudate present during ecdysis of H. contortus L3. Antibody 8D.3 reacted with the SC of these nematodes on cryosections but on live nematodes the immunofluorescence was very patchy and was shed from the nematode SC.

Infection with the cestode Spirometra spp. was studied in 2 populations of lions in the Serengeti and the Ngorongoro Crater in Tanzania, East Africa. These 2 lion populations lived in different habitats and were known to differ genetically: lions in the Serengeti were outbred, whereas lions in the Ngorongoro Crater were inbred. Faecal samples were collected from 112 individually known lions between March 1991 and November 1992. Over 60% of lions were infected and the median intensity of infection was 975 eggs per g of faeces. The distribution of egg counts was overdispersed. There was variability through time, though this was unrelated to seasons delimited by rainfall. There were no significant differences in levels of infection between age classes; cubs less than 9 months were already heavily infected. Sex and reproductive status did not have a significant effect. However, there were significant differences in intensities of infection between the Crater and the Serengeti populations – Spirometra spp. showed a higher level of infection intensity in the Crater population – with some variation between prides within these populations. Allozyme heterozygosity scores were available for a subset of 28 lions but were unrelated to levels of Spirometra infection. It was not possible to ascribe differences in levels of parasite infection to genetic rather than ecological factors.

Patterns of nematode fecundity were investigated for infections of the caecal worm Heterakis gallinarum in the ring-necked pheasant (Phasianus colchicus). Worm length was a good predictor of parasite fecundity. After controlling for worm length no other factors, including parasite intensity, were related to worm fecundity. Density dependence in worm size was detected in natural infections at parasite intensities above a threshold of 96 worms (worm size decreased with increasing parasite intensity). However, below this threshold, worm size actually decreased with decreasing parasite intensity (inverse density dependence). The interaction between density dependence and inverse density dependence in regulating the development and subsequent fecundity of H. gallinarum worms in ring-necked pheasants was demonstrated in an infection experiment. Density dependence was observed in the stunted growth of worms in heavily infected hosts, relative to worms in lightly infected hosts. Inverse density dependence in worm size was the common pattern across hosts by the end of the experiment, when parasite intensities were below the density dependence threshold. This is the first study to document both density dependence and inverse density dependence in parasite fecundity in the same host–helminth system.

The epidemiology and genetic variability of 2 parasitic nematodes Heligmosomoides polygyrus and Syphacia stroma of Apodemus spp. were investigated. Both are parasites of the same host, exhibit a direct life-cycle and are dioecious. However, H. polygyrus has a diploid and S. stroma a haplodiploid mode of reproduction. Haplodiploidy may lead to a more female biased sex ratio and reduced genetic variability. Levels of genetic diversity were analysed using both isoenzyme electrophoresis and RAPDs (random amplified polymorphic DNA). Both parasites showed a female biased sex ratio with a stronger bias for the haplodiploid parasite. Results showed significantly fewer genetic polymorphisms as measured by RAPDs for the haplodiploid parasite S. stroma in comparison with H. polygyrus. Despite the observed female biased sex ratio this could not be explained by a significant amount of inbreeding. Heterozygote deficiency for individual allozyme loci – which could indicate inbreeding – was not found in either parasite species. Other features of the particular life-history of these species are likely to have an impact on the sex ratio and genetic variability too.

The monogenean (platyhelminth) skin parasite Entobdella soleae from the common sole (Solea solea) lays tetrahedral eggs. One of the 4 corners of the tetrahedron is a detachable operculum which is bonded to the rest of the egg-shell by cement. Most of this cement layer, beginning at the inner surface of the shell and running through almost to the outer surface (a distance of about 2 μm), is more or less uniform in thickness (30–38 nm), or tapers slightly. About 345 nm from the outer surface the cement layer narrows abruptly to about 10 nm. The cement is exposed on the inner surface of the shell, but in most eggs a layer of shell about 10 nm thick covers the narrow outer region of the cement layer. When experimentally perforated eggs were incubated with trypsin, the wide inner layer of cement was digested, but the narrow outer region initially remained intact. These observations are discussed in relation to the following (1) survival of the eggs during embryonic development, (2) hatching, (3) the ‘hinge’ often connecting the operculum to the empty egg-shell, (4) the rapid hatching that occurs in some other monogeneans.