A microsporidium of the genus Encephalitozoon was isolated into culture from the nasal epithelium of a patient with AIDS. It was compared with in vitro isolates of Encephalitozoon cuniculi and the type isolate of Encephalitozoon hellem by SDS–PAGE and by Western blotting with murine antisera raised to E. cuniculi, E. hellem and the nasal isolate, monoclonal antibodies raised to E. cuniculi and sequential sera from the patient. All tests showed similarities between E. hellem and the nasal isolate but differences between these two isolates and E. cuniculi. Minor protein differences between E. hellem and the nasal isolate were not considered sufficient to separate them at the specific level. The new isolate is named the Wainwright isolate of E. hellem. The ultrastructure of the Wainwright isolate in vitro was similar to that of the parasite in vivo but there was a greater tendency for disruption of the parasitophorous vacuoles. The deposition of the electrondense surface coat on the sporogonic stages of E. hellem, as a uniform layer which later thickens, is in contrast to its deposition as broad bands, which later join up, in E. cuniculi. This may be a useful character in distinguishing the species without recourse to analysis of protein profiles.

Adhesion of parasitized red blood cells to vascular endothelium is thought to play an important role in the development of the ischaemic complications associated with severe falciparum malaria. Using a novel, flow-based assay, we have investigated the adhesion of parasitized red blood cells to formalin-fixed human umbilical vein endothelial cells (HUVEC), for isolates obtained from 32 Gambian subjects with mild or severe falciparum malaria. Red cells infected with wild strains of Plasmodium falciparum were able to adhere to HUVEC under physiologically relevant flow conditions, but the level of adhesion was highly variable, ranging from 1 to 688 adherent cells per mm2 of HUVEC. Within isolates, some adherent parasitized cells remained stationary, whilst other formed less stable interactions and rolled slowly over the cell surface. There was no significant difference in adhesion of parasitized cells between isolates obtained from mild or severe cases of malaria, although a subset of isolates did show very high levels of adhesion. The results suggest that there is not a simple relationship between the adhesion of parasitized cells to cultured endothelial cells (presumably via the receptor ICAM-1) and the clinical severity of the disease, although variation in microvascular adhesion in vivo may still be a determinant of ischaemic complications.

The gene for the 32 kDa surface protein (p32) of Theileria sergenti was cloned into λgt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected the Pst I-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and that transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

Malate dehydrogenase (decarboxylating) from Tritrichomonas foetus hydrogenosomes was purified close to homogeneity by a combination of differential centrifugation, zwitterionic detergent solubilization, Red-Sepharose chromatography and anion-exchange chromatography. The enzyme with apparent subunit size of 59 kDa and native molecular mass of 308 kDa utilized NAD+ preferentially to NADP+ as a cofactor and required Mn2+ or Mg2+ for its activity. Affinity curves for malate and coenzymes were hyperbolic. Km for malate was 100 μM and 458 μM in the presence of NAD+ and NADP+, respectively. Km for NAD+ and for NADP+ in the presence of malate was 18 μM and 207 μM, respectively. The enzyme is proposed to be a tetramer with a possible physiological role in the maintenance of an appropriate NAD+/NADH ratio in hydrogenosomes.

The nature of stimulus-induced flaccid paralysis produced in Mg2+-paralysed Schistosoma mansoni was investigated. Serotonin induced a dose-dependent, heterologous flaccid paralysis with an IC50 of 600 nm. This flaccid paralysis was a function of the extracellular Mg2+:Ca2+ ratio and was reversible. Tonic contractions produced by phorbol-12,13- dibutyrate or 60 mM K+ were reversed by the application of serotonin and flaccid paralysis was induced. These actions of serotonin were mimicked by forskolin and synergized by IBMX but the potassium channel blocker, 3,4-DAP, did not produce flaccid paralysis. When Mg2+-paralysed parasites were stimulated with 3,4-DAP, IBMX produced a dose- dependent flaccid paralysis with an IC50 of 11 μm. Membrane permeable analogues of cAMP and cGMP did not synergize with IBMX. Cholinergic agonists, but not other inhibitory substances, prevented the serotonin- and forskolin-induced and the IBMX-synergized flaccid paralysis but not that produced by praziquantel. The possible interactions of these agents with the muscle are discussed.

In the present study fibrogenic gene expression was determined in murine Schistosoma japonicum infection during the progression of immune modulation of infection and following chemotherapy during the course of immune modulation. Histomorphometric analysis of granuloma size and collagen deposition revealed peak granuloma size in acute infection (5 weeks) and peak hepatic collagen content at 16 weeks of infection. Peak Type I collagen gene expression was concomitant with TGF-β1 gene expression at 8–11 weeks. Chemotherapy during either acute (9 weeks) or chronic (24, 28 weeks) infection resulted in increased collagen deposition and increased gene expression of Type I collagen and TGF-β1. However, chemotherapy at 14–16 weeks resulted in decreased levels of TGF-β1 gene expression and essentially minimal change in Type I collagen deposition and gene expression. These data indicate that chemotherapy of schistosomiasis japonica does not reverse hepatic fibrogenesis when administered in acute infection – when granuloma size is maximal – or in chronic infection. However, a beneficial effect on hepatic fibrogenesis is seen when chemotherapy is administered at 14–16 weeks post-infection, a time of decreasing granuloma size and maximal hepatic collagen content. Thus the ability to reverse schistosomal-induced hepatic fibrogenesis by chemotherapy may depend on disease stage.

Isolates of Trypanosoma cruzi from human patients, domestic and sylvatic animals and vector insects were obtained in different areas of Argentina. Electrophoretic patterns of enzymes from extracts of 95 isolates were analysed. On the basis of zymograms providing information on 10 loci, 12 zymodemes are described according to their genotypes. Data presented show fixed heterozygosity, absence of segregation of genotypes, significant departures from Hardy–Weinberg equilibrium, and over-represented genotypes. This evidence supports the hypothesis that sexual reproduction is very restricted or absent in this parasite. The proportion of polymorphic loci is 80%. The expected mean heterozygosity per locus (He) is 0·43, while the observed value (Ho) is 0·24. Differences between these values may be explained by accepting a basically clonal structure for T. cruzi. The data matrix of 12 zymodemes using 28 characters was analysed using a Wagner parsimony algorithm. Two equally most parsimonious unrooted trees were generated; both have 39 steps. The results show clusters clearly separated according to the geographical origin of the stocks. There are some indications of some correlations between genetic composition of the parasite and the clinical picture of the infection in human patients.

Serum from uninfected mice of different strains, as well as from germ-free animals, contains antibodies which react specifically with at least two trypanosomal proteins, I/6 and MARP1. These antibody populations are highly specific for the respective proteins, are of similar affinity as hyperimmune antibodies, and consist of IgM as well as IgG isotypes. Hyperimmune antibody raised against the cross-reacting trypanosomal protein I/6 detects a 60 kDa protein in mouse 3T6 cells, which is a component of the fibroblast cytoskeleton.

The random amplified polymorphic DNA (RAPD) technique was successfully used to produce genetic fingerprints distinguishing between Trichinella spiralis and Trichinella britovi. The same patterns were obtained from purified and crude DNA preparations of pooled and single muscle larvae. RAPD fingerprinting was applied to muscle larvae preserved under different conditions and recovered from different hosts. Larvae recovered from fresh and frozen meat and stored at – 20 °C for a long time or under 70% ethyl alcohol at room temperature for 30 d gave good and reproducible results. Single larvae recovered from a naturally infected wild boar and from a human biopsy gave fingerprints congruent to those obtained from T. britovi reference strains. The results prove that RAPD analysis is a quick method to distinguish between the autochthonous Trichinella species of Central-Southern Europe in less than 1 d after the detection of the infection. If necessary, the biological material can be frozen or stored under 70% ethyl alcohol at room temperature and sent to laboratories able to perform the RAPD analysis. The RAPD technique requires no prior knowledge of the molecular biology of the organism to be investigated and therefore appears to be a promising tool in parasitology for the identification of sibling species.

The course of a primary Necator americanus infection was studied in the lungs and small intestines of syngeneic mice. Following percutaneous infection no difference in initial larval establishment in the lungs was found between male BALB/c, NIH or B10.G mice. However, significant differences in the subsequent kinetics of infection were demonstrated between the BALB/c and NIH strains. Lung worm burdens declined more slowly in NIH mice than in BALB/c strain. Surprisingly, however, a greater proportion of larvae remaining in the lungs of BALB/c mice, 9 days p.i., were trapped than in NIH mice. Nevertheless, establishment in the small intestines of the BALB/c strain was consistently greater than in NIH mice. Host immunosuppression resulted in increased larval retention in the lungs of both the BALB/c and NIH strains as well as in the small intestines of BALB/c mice. Treatment with hydrocortisone acetate did not increase intestinal worm burdens in NIH mice. The data presented suggest that, in this complex, dynamic model system, designation of ‘susceptible’ and ‘resistant’ strains is inappropriate. The factors underlying the observed strain differences in resistance to infection are discussed.

Freshly isolated pronephric leucocytes from roach, Rutilus rutilus and gudgeon, Gobio gobio were exposed to extracts of plerocercoids of Ligula intestinalis from these two cyprinid fish. Addition of the extracts or an increase in incubation temperature from 10 to 20 °C induced polarization of neutrophils and L1 granulocytes. Cells were transformed from their typical spherical shape to elongate forms possessing a ruffled leading edge. Extracts obtained from gudgeon-Ligula stimulated polarization of both roach and gudgeon leucocytes at 10 and 20 °C. In contrast, extracts from roach-Ligula, whilst having little effect at 10 °C, suppressed temperature-induced polarization of leucocytes at 20 °C. Addition of serum to all the essays enhanced polarization and abolished the roach-Ligula-induced suppression. It is suggested that leucocyte chemoattractants are present in Ligula from roach and gudgeon and only parasites from the former host contain an inhibitor of polarization. In addition, host-derived factors possibly complement, may be involved in leucocyte chemoattraction.

Sphaeridiotrema pseudoglobulus is a digenean parasite believed to be involved in a yearly fall die-off of ducks in Québec, Canada. Hatching characteristics of eggs stored at 7°C for 0–28 weeks in the lab and following maintenance overwinter in a lake are described. The hatching success of eggs stored for 4–28 weeks remained constant (71–81 %) but slightly less than that observed in fresh eggs (90%). The hatching success of eggs kept overwinter under natural conditions did not differ from that of eggs stored an equivalent length of time in the lab at 7°C (74·7 and 75·8%, respectively). With the exception of fresh eggs (17·7 days), the mean hatch time of eggs steadily decreased with increased storage time (18·9 days following 4 weeks storage to 11·4 days at 28 weeks storage) due to a slow embryonation of the eggs at 7°C. Hatching characteristics of a subsample of eggs incubated at 10, 15 and 20°C were compared and the embryonation rate was found to increase with incubation temperature. The majority of eggs stored at 10°C embryonated but failed to hatch. When their incubation temperature was raised to 15°C, a further 46% hatched within the following week. The survivorship functions of miracidia hatching from eggs stored for 8, 12, 16 and 20 weeks differed but the mean expected life-span of the miracidia did not decline with increasing storage time as expected. The results of these experiments are discussed in relation to the potential importance of overwintered eggs in the development of the infective pool of metacercariae.

The expression of a protein complex designated gp15/400, previously identified via extrinsic iodination of adult Brugia malayi, was examined by labelling all stages found in the mammalian host and immunoprecipitation with a specific antibody raised to a recombinant protein. In this way, gp15/400 could be detected in L3, L4, adult worms and microfilariae recovered from jirds and labelled with Bolton–Hunter reagent. Metabolic labelling indicated that gp15/400 was released into culture medium when adult worms were maintained in vitro, but at a rate slower than that of gp29, the major soluble cuticular glycoprotein. Immuno-electron microscopy showed that the protein complex was broadly distributed in different tissues, although it was not detectable in the cuticle of adult worms. Dense labelling was observed in the matrix of the basal laminae bordering the hypodermis, somatic musculature and oesophagus, and lower but significant labelling was seen in the cells overlying these extracellular matrices. Hybridization of genomic DNA with a cDNA probe encoding gp15/400 indicated that homologous genes were present in Dirofilaria immitis and Acanthocheilonema viteae. The failure to detect related genes in non-filarial nematodes was presumed to be due to divergence beyond the practical limits of detection by nucleic acid probes, as antibody reagents showed that the protein cross-reacted immunologically with ABA-1, a major protein allergen from the body fluid of Ascaris.

The effects of the insect hormones, ecdysone and 20-hydroxyecdysone, certain non-steroidal ecdysteroid agonists (RH compounds) and the inhibitor, azadirachtin, on the timing of the 3rd-stage moult of Dirofilaria immitis were investigated. 20-Hydroxyecdysone and RH 5849 when used at a concentration of 10−5 M, resulted in a premature timing of this moult. Azadiracthin, at a similar concentration, prevented moulting of most of the larvae to the 4th stage. The results are discussed in relation to the possibility of a hormonal role for ecdysteroids and neuropeptide-like compounds in the control of ecdysis in filarial nematodes, that maybe somewhat comparable to the system which is found in insects.