Experiments were designed to look at the relationship between infective dose and disease severity using 2 clones of Plasmodium chabaudi that differ in virulence. We asked whether there were dose–severity relationships, whether clone differences in virulence were maintained over a range of doses, and whether disease severity could be accounted for by parasite dynamics. Groups of mice were infected with parasite doses differing by an order of magnitude, ranging from 100 to 1×108 parasites. Infective dose affected the probability of death, but only with the more virulent clone. Dose also affected morbidity. For both clones, higher doses induced greater anaemia. Larger doses caused greater weight loss, but only for infections with the more virulent clone. Here, for a given dose, mice lost a fixed amount of weight, irrespective of their initial weight. Larger doses induced earlier mortality and morbidity than did lower dose treatments. Finally, dose affected parasite dynamics, with earlier and higher peak parasite densities in larger dose infections. All these effects were small relative to clone differences in disease severity, which were apparent across the range of doses. Dose effects were manifested through the timing and/or magnitude of peak parasite densities, broadly supporting the idea that dose affects disease severity by altering the time the host has to control parasite densities and ameliorate the effects of parasites. We discuss the possible efficacy of intervention strategies aimed at reducing human disease severity by reducing infective parasite dose.

Neospora caninum is a protozoan parasite that causes bovine abortion. The epidemiology of N.caninum is poorly understood and little is known about the genetic diversity of the parasite, or whether individual isolates differ in virulence. Such diversity may, among other factors, underlie the range of pathologies seen in cattle. In this study we analysed biological and genetic variation in 6 isolates of N. caninum originating from canine and bovine hosts by measurement of growth rate in vitro, Western blotting and random amplification of polymorphic DNA (RAPD). This comparative analysis of intra-species diversity demonstrated that heterogeneity exists within the species. The relative growth rate in vitro, as assessed by 3[H]uracil uptake, showed significant variation between isolates. However, no significant differences were detected between the antigenic profiles of each isolate by Western blotting. RAPD-PCR was performed on DNA from the 6 Neospora isolates; 3 strains of Toxoplasma gondii, Sarcocystis sp. and Cryptosporidium parvum were also analysed. Twenty-six RAPD primers gave rise to 434 markers of which 222 were conserved between all the Neospora isolates and distinguished them from the other Apicomplexa. An additional 54 markers were unique for Neospora but were polymorphic within the species and able to differentiate between the individual isolates. The RAPD data were subjected to pair-wise similarity and cluster analysis and showed that the Neospora isolates clustered together as a group, with T. gondii as their nearest neighbour. N. caninum isolates showed no clustering with respect either to host or geographical origin. The genetic similarity between Neospora isolates from cattle and dogs suggests that these hosts may be epidemiologically related, although further analysis of bovine and canine field samples are required. The genetic and biological diversity observed in this study may have important implications for our understanding of the pathology and epidemiology of neosporosis.

Interferon-gamma (IFN-γ) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-γ affects the parasite-infected host cells. The effect of IFN-γ on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-γ, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-γ. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-γ-treated cells following N. caninum infection. Moreover, IFN-γ treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-γ induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the β-galactosyltransferase (β-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-α-N-acetyl-glucosaminyltransferase (O-α-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the β-Gal transferase, the O-α-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Susceptibility to oxygen and properties relative to oxygen metabolism were compared in metronidazole-resistant and susceptible strains of Trichomonas vaginalis. The study involved clinical isolates displaying the aerobic type of resistance, as well as resistant strains developed in vitro, both with aerobic (MR-3) and anaerobic (MR-5, MR-100) resistance. Elevated sensitivity to oxygen of the resistant clinical isolates was observed. Progressive increase of susceptibility to oxygen also accompanied in vitro development of resistance. No correlation was found between the activity of NADH oxidase and aerobic resistance, while the in vitro derivative with fully developed anaerobic resistance (MR-100) showed about 50% decrease of NADH oxidase activity. The superoxide dismutase (SOD) activity was elevated in both resistant clinical isolates and in in vitro-developed resistant strains. The changes in levels of ferredoxin were insufficient to support ferredoxin deficiency as a cause of aerobic metronidazole resistance. Western blot analysis and electron paramagnetic resonance spectroscopy of purified hydrogenosomes showed that ferredoxin is expressed in aerobically resistant strains and has intact iron-sulfur clusters. Down-regulation of ferredoxin was demonstrated only in the late phase of development of the anaerobic resistance (MR-100). The results support a link between aerobic resistance and defective oxygen scavenging. The increased levels of intracellular oxygen, beneficial to resistant parasites when they interact with the drug, may have adverse effects on their fitness as shown by their increased sensitivity to oxidative stress.

Antimicrobial proteins were purified from acid extracts of rainbow trout (Oncorhynchus mykiss) and sunshine bass (Morone saxatilis male×M. chrysops female) skin, gill and spleen by reverse-phase HPLC. Mass spectrometry and amino acid sequence data suggest that these proteins are closely related to histone H2B and histone H1 and thus they were designated histone-like proteins (HLPs). These proteins were lethal to Amyloodinium ocellatum, which is one of the most important parasitic agents affecting fish. Antibiotic concentrations as low as 12·5 μg/ml were inhibitory. Activity was directed against the trophont (feeding) stage of the parasite, while the disseminative (dinospore) stage was unaffected. Thus, HLPs act unlike typical drugs used to treat amyloodiniosis, which usually target the dinospore. Both the ability of the parasite to infect host cells, as well as the ability to grow and differentiate after infection were severely inhibited. This is in contrast to magainin 2, which was similarly toxic to both the dinospore and trophont stages. These findings provide further evidence that histone-like proteins may be important defensive molecules in fish.

The avian schistosomatid Trichobilharzia ocellata plays an important role as causative agent of cercarial dermatitis of humans in Europe. In order to improve the taxonomic knowledge on this parasite, studies of miracidial chemo-orientation as well as experimental infections of different snail species were conducted using strains of T. ocellata and T. franki. Both schistosomes exhibited a high intermediate host specificity. The miracidia clearly preferred the SCW (snail-conditioned water) of the respective natural intermediate host to SCW of other sympatric snail species. T. ocellata proved to be capable of infecting Lymnaea stagnalis and Stagnicola palustris, but could not develop in Radix ovata or R. auricularia. T. franki established an infection in specimens of R. auricularia and R. ovata, but not in L. stagnalis or S. palustris. The results imply that the intermediate host spectrum of T. ocellata is limited to L. stagnalis and S. palustris. Findings of T. ocellata (or Cercaria ocellata) that originated from snails of the genus Radix are likely to have actually belonged to species such as T. franki or T. regenti. The assumption that T. szidati is synonymous to T. ocellata is also discussed.

In order to identify antigens associated with protection and those associated with active infection, the humoral immune response of 6 Mandrillus sphinx immunized with 150 irradiated L3 and challenged with 100 normal L3 of Loa loa or 6 animals infected with 100 L3 were compared. The plasma of these animals was analysed by Western blot using adult, Mf and L3 antigens. Several antigens with molecular weights varying from 120 kDa to 13 kDa were recognized by the plasma of all animals. It was shown that early recognition of microfilarial antigens with molecular weights of 97, 68, 45 and 33 kDa correlated with the amicrofilaraemic state. A total of 83% of animals with circulating microfilariae had antibodies against the microfilariae 21 kDa antigen. Furthermore, the antibodies against the 21 kDa appeared 1 month before detection of microfilariae in the peripheral blood of 80% of these animals, and declined when animals became amicrofilaraemic. In contrast, when L3 antigen was used, a molecule with a relative molecular weight of 20 kDa was recognized by antibodies of the only animal which remained amicrofilaraemic for 1 year after immunization with irradiated L3. These results suggest that the microfilarial molecule of 21 kDa may be useful as a marker of Loa loa patent infection, whereas the 97, 68, 45 and 33 kDa molecules of microfilariae and the L3 molecule of 20 kDa may be associated with resistance against Loa loa.

We produced a recombinant protein from a cDNA library from muscle larvae of Trichinella spiralis which had proteinase inhibitory activity. The predicted amino acid sequence of the clone had an identity of only 30% to the serine proteinase inhibitors (serpins) from Caenorhabditis elegans or Brugia malayi. At the putative reactive region, however, the identity was about 50%. The recombinant protein expressed in Escherichia coli inhibited 82% of the activity of the serine proteinase (trypsin). Stage-specific expression of this protein was suggested from the following experiments. Antibody against the recombinant protein could stain proteins migrating at about 42 kDa (which is the expected size from the sequence) in crude extracts from newborn larvae and 18-day post-infection (p.i.) muscle larvae, but it failed to stain any proteins in crude extracts from 30-day p.i. muscle larvae. Production of mRNA transcript for the serpin gene was restricted largely to the newborn larvae and to 18-day p.i. muscle larvae. The antibody reacted with the stichocytes of the larvae at 18 days p.i., but did not react with the muscle larvae at 24 days and 30 days p.i.

Infection of an intracellular parasitic nematode, Trichinella spiralis, resulted in severe damage in muscle cells which was followed by activation and proliferation of satellite cells. The repairing process, shortly after the damage, histopathologically resembled those seen after mechanical injury. Resemblance was also true for kinetics of expression of myogenic regulatory factors (MyoD, myogenin and MRF4). The difference resided in the next step where the muscle cell infected with T. spiralis transformed to a unique cell which is parasitologically known as the nurse cell, and the proliferated satellite cells did not differentiate to the muscle cell but to the nurse cell (misdifferentiation). Thus the nurse cell was a fusion of the transformed infected muscle cell and misdifferentiated satellite cells. Infection with another species of Trichinella, T. pseudospiralis, also caused cell damage, but more extensively involving the entire length of the infected muscle cells because no septum was formed to minimize the affected area. Therefore, a large number of satellite cells were activated and proliferated. The myogenic regulatory factors such as MyoD and myogenin were activated for a longer period than in the case with T. spiralis infection. The infected muscle cell transformed to the nurse cell, whose cytoplasm was characterized by extensive smooth endoplasmic reticulum. Satellite cells misdifferentiated to the nurse cell, whose cytoplasm was amorphous, void of distinct cell organelles. The two kinds of cytoplasm did not fuse as examined thus far. Thus infection with T. spiralis and T. pseudospiralis caused misdifferentiation of satellite cells, but in a different way.

Strong resistance to reinfection developed in pigs as a result of trickle inoculations with Trichuris suis (250 infective eggs twice weekly for 4 weeks). At challenge (4000 eggs) 8 weeks after the last trickle inoculation, we observed a 96% reduction in the mean number of established challenge worms in immunized (TRI/CHA) compared with non-immunized controls (CON/CHA) (74 v. 1753; P = 0·004). Moreover, the total faecal egg excretion was significantly lower in immunized pigs compared with controls (P = 0·0003). The trickle inoculations depressed pig weight gains over the 8-week course of immunization (P = 0·042). It was observed that a small number of pigs developed a weaker level of resistance, reflecting the importance of genetic determinants. The relevance of these data to the population biology of porcine and human whipworm infections is discussed.

This experiment was designed to determine the effects of under-dosing on the frequency of benzimidazole resistant allele in the nematode Teladorsagia circumcincta. Fenbendazole (FBZ) was tested at 1/32, 1/16, 1/8 and 1/4 of the recommended dose for sheep (5 mg/kg body weight). The fraction of the susceptible homozygote (SS), susceptible heterozygote (RS) and resistant homozygote (RR) genotypes were compared among FBZ dose groups to evaluate differences between SS and RS genotype selective advantage. Almost all SS genotype worms were eliminated by 1/4 of the FBZ recommended dose, whereas a significant fraction of the RS genotype worms survived treatment. The selective advantage was 4·5 times higher for the RS genotype. This selective advantage was determined at 1/4 of the manufacturer's recommended dose of FBZ. This value should be taken as an indictor of the selective advantage of RS over the SS genotype when lambs are under-dosed. A computer simulation was used to study the putative spread of anthelmintic resistance over a range of RS selective advantages (2, 4·5 and 10-fold), with two average sizes of individual host worm population (20 or 2000 worms/host) and two initial R allele frequencies (0·1% or 1%). In all situations, the lowest selective advantage of the RS genotype over the SS genotype was sufficient to promote the spread of resistance in susceptible populations. When the RS genotype had no selective advantage over the SS genotype, genetic drift almost always led to the loss of the R allele, except in the largest populations (average size = 2000 worms).