The prevailing hypothesis concerning the pathogenesis of toxoplasmic hydrocephalus alleges that (a) parasites invade and destroy the ependymal lining of the lateral ventricles, followed by (b) the sloughing of masses of degenerating ependymal and inflammatory cells leading to obstruction of the ventricular foramina and aqueduct of Sylvius, thereby initiating the hydrocephalus. Our observations in chronically infected mice indicate otherwise. Parasite invasion of the ependyma was not detected; the intraventricular masses of cellular ‘debris’ contained neither ependymal nor inflammatory cells; and obstruction of the ventricular foramina and/or aqueduct was not seen. As an alternative hypothesis, we suggest the development of hydrocephalus in the infected mice was consequent to severe leptomeningeal inflammation blocking the subarachnoid space and impeding the resorption of cerebrospinal fluid by the arachnoid villi. Narrowing of the aqueduct of Sylvius, when present, was adjudged the result, not the cause of the hydrocephalus, due to compression of the midbrain by the enlarging lateral ventricles.

The venerid clams, Tapes decussatus and T. semidecussatus, develop a singular defensive response to Perkinsus atlanticus infection. This reaction involves the redifferentiation of recruited granulocytes and the expression de novo of the polypeptide p225. To determine whether the association of this defensive process with the natural parasitism by P. atlanticus is unique, the inflammatory response elicited by inoculations of bacteria, algae and non-viable P. atlanticus pre-zoosporangia in the clam T. semidecussatus was shown. Inoculated areas were heavily infiltrated by granulocytes and delimited by myofibroblast-like cells and extracellular matrix. While bacteria and algae were phagocytosed by the infiltrated granulocytes, pre-zoosporangia were not. After 40 days, neither cell redifferentiation nor the expression of p225 was observed. These findings indicate that both redifferentiation and p225 expression are specifically associated with P. atlanticus infection. After 5-bromodeoxyuridine administration, only a few cells were labelled either in the inoculated zone or in the cellular reaction around P. atlanticus meronts. Significant differences between untreated and inoculated groups were observed in the epicardic connective tissue soon after injection. These results suggest that this anatomical region could be the main site of haemocyte proliferation stimulated after inoculation of foreign bodies in T. semidecussatus.

Hypoxanthine transport in the insect trypanosome, Crithidia luciliae, was activated in purine depleted conditions. The existence of 2 hypoxanthine transport mechanisms was established. The first, a non-saturable diffusion system, present in purine replete conditions, exhibited properties that were different from the second transport system which was evident only during purine depleted conditions (purine stress). The rate of transport under purine stress was elevated approximately 8-fold over that in replete conditions. This transporter was saturable with a Km of 3·9 μM for hypoxanthine. The transporter substrate specificity included other purine bases, e.g. adenine and guanine, and the purine nucleoside, adenosine. These inhibited hypoxanthine transport competitively with Ki values of 2 μM, 3 μM and 42 μM respectively. Coincident with the increase of hypoxanthine transport under purine stress, the transport of adenosine increased 4-fold and the activity of the 3′-nucleotidase ectoenzyme also increased significantly. Under purine stress the concurrent increase of hypoxanthine and adenosine transport and the increase in 3′-nucleotidase activity could be repressed with either the supplementation of excess purines or by cycloheximide. This study of purine salvage mechanisms in Crithidia luciliae illustrates the successful adaptation of the parasite to nutritional insufficiency.

This is a report of the prevalence, transmission and intensity of infection of a microsporidian sex ratio distorter in natural populations of its crustacean host Gammarus duebeni. Prevalence in the adult host population reflects differences in the intensity of infection in transovarially infected embryos and in adult gonadal tissue. The efficiency of transovarial parasite transmission to young also differs between populations, but this alone is insufficient to explain observed patterns of prevalence. Infection intensity may be important in determining future infection of target tissue in the adult and subsequent transmission to future host generations. We consider patterns of parasite infection in terms of selection on transmission and virulence.

Developmental rates for Wuchereria bancrofti larvae maturing in the vector Aedes polynesiensis were estimated by analysing stage-frequency data consisting of counts of larval stages in mosquitoes reared at 20, 22·5, 25, 27·5, 30 and 32 °C. Base temperatures (i.e. low temperature thresholds) for W. bancrofti development were estimated by the x-intercept method and the model of Lactin et al. (1995). Resulting values were similar with both methods and were ≈12·5 °C for microfilariae (mf) in thorax, ≈17 °C for L1, 15·5 °C for L2 and 16·5 °C for L3. Upper thresholds estimated by the Lactin et al. model were 29·3 °C for mf, 29·1 °C for L1, 32·2 °C for L2 and 31·5 °C for L3. In addition, an original method was devised for computing the L3 upper threshold, by modelling L3 length shrinkage with temperature. It gave a value of 31·4 °C. At 32 °C, L2 and L3 stages exhibited altered morphology, larvae being shorter and wider than expected. The model of Lactin et al. described adequately the non-linear relationship between developmental rate and temperature, but a linear degree-day approach may be applied for temperatures below 27–28 °C.

Molecular cloning and immunoelectron microscopy have been used to clone the full-length gene encoding Cryptosporidium parvum oocyst wall protein (COWP) and to analyse at the ultrastructural level the expression and localization of COWP during development in the gut. COWP is 1622 amino acids long, has a typical leader peptide and consists of 2 amino acidic domains each containing distinct repeated elements possibly originating from a common ancestral precursor. Electron microscopy localized COWP in a large cytoplasmic inclusion and in the wall-forming bodies of early and late macrogametes, respectively. Ultrastructural analysis of double-walled sporulating and mature oocysts indicated that COWP is selectively localized in the inner layer of the oocyst wall. This study provides the first localization at the ultrastructural level of a cloned coccidian oocyst wall protein.

We have used a chromosome-specific approach to generate a 300 kb long ‘contig’ across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The ‘contig’ generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb ‘contig’ rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.

The enzymes and receptors in parasites that can be qualified as targets for antiparasite chemotherapy should perform essential functions in the parasites and demonstrate some feasibility for selective inhibition. They can be tentatively identified through detailed analysis of various aspects of metabolisms in the parasites or elucidation of the mechanisms of action among proven antiparasitic agents. Preliminary verifications of these putative targets can be indicated by in vitro antiparasite activity of an inhibitor of the target. However, before a major long-term effort to pursue in-depth structure-activity analysis of the target is to be committed for specific inhibitor design, further validations of the target are essential to insure that future studies are not misguided. One old-fashioned approach to validate a target in the pharmaceutical industry is by correlating target inhibitions with antiparasitic activities among large numbers of drug derivatives. The results are often indicative but hardly ever conclusive. Another method is by comparing the putative drug targets between the drug-sensitive and the drug-resistant parasites for potential discrepancies. Unfortunately, the latter often result from indirect causes, such as reduced drug transport, instead of an alteration of the drug target itself. The third experimental approach is by disrupting the gene encoding the putative target in parasite, which can provide the most conclusive evidence on whether the target plays an indispensible role in the parasite. But special conditions are needed for the gene knockout mutants to survive to exhibit their phenotypes and to allow genetic complementation studies for further verifications. Furthermore, gene knockout experiments are often difficult to perform on cells of multiple ploidy or genes of multiple copies, and are currently applicable only to a limited number of protozoan parasites. In the current article I have tried to take a cursory look at some eleven putative drug targets among various parasites, each supported by well-established antiparasitic agents identified as its inhibitors. I have also considered the evidence for validity of each of them and the potential means of further verifying their validity.

Experimental exposure to bleached kraft pulp and paper mill effluent (BKME) at a concentration of 10% significantly reduced the abundance and mean number of species of Dactylogyrus on the gills of naturally infected roach, Rutilus rutilus, over a 3 week period. Seven Dactylogyrus species were recorded which differed widely in their susceptibility to the effluent. The experiment coincided with a natural spring peak in dactylogyrid infections on roach. BMKE exposure did not prevent parasite reproduction but post-larval abundance was significantly higher on control than effluent-exposed hosts. There was no evidence of a BKME-induced shift in microhabitat distribution of dactylogyrids. Elevated levels of infection with Dactylogyrus spp. have been recorded on roach from a Finnish lake containing relatively low concentrations of BMKE. The high exposure concentration in this experiment produced a similar reduction in dactylogyrid infections to that reported in separate studies in close proximity to Swedish pulp and paper mills. The discrepancy between the results of the Finnish and Swedish field investigations is therefore considered to be due to differences in BKME concentration between the study areas.

The microsporidian Encephalitozoon cuniculi can infect a wide variety of mammals including man. In this study, E. cuniculi isolates of animal origin were compared with 6 isolates obtained from HIV-infected patients. Based on results of Western blot analysis, random amplified polymorphic DNA (RAPD) and the sequence of the rDNA internal transcribed spacer (ITS) the isolates were classified into 3 groups with the repeated element 5′GTTT-3′ in the ITS being a reliable genetic marker. Five isolates from Swiss patients were found to be homologous to isolates from Swiss rabbits (strain I). The sixth isolate from a patient from Mexico differed by all methods and could be attributed to E. cuniculi strain III that has been described from 2 dogs from the USA. All of these isolates were distinguished from isolates from blue foxes from Norway (strain II). Intraspecific nucleotide divergence of the SSU rRNA gene of E. cuniculi belonging to the 3 strains was in the same low range (0·00–0·15%) as was found for the corresponding sequence of 2 E. hellem isolates. Groups of 2 rabbits were infected by oral inoculation of 107E. cuniculi spores (2 isolates from strain I of human and rabbit origin, 1 from strain III) as shown by antibody responses and the re-isolation of the parasites from brain material. The results provide further evidence that per oral transmission of the parasite between various hosts is feasible.

Chemically-modified oligonucleotides are now routinely used to prevent gene expression in cell-free media and in cultured cells. The binding of an antisense sequence to a complementary RNA target may lead to the selective inhibition of the encoded information. This may occur at different levels: splicing; transport of the mature RNA from the nucleus to the cytoplasm; translation. Antisense oligonucleotides constitute an interesting tool to shed some light on gene function. They are also potential new therapeutic agents against pathogenic organisms. This review discusses the rules that guide the design of an antisense oligomer and the choice of a target sequence. Examples of the potential use of antisense oligonucleotides in the fields of virology and parasitology, in particular in relation to trypanosomatids, are described.

Excretory–secretory (E–S) products, released by Trichobilharzia ocellata cultured in vitro, were assessed for their modulatory effects on the internal defence system of the freshwater snail, Lymnaea stagnalis. E–S products were concentrated and fractionated using high-pressure liquid chromatography (HPLC) and bacterial clearance activity of haemocytes was taken as a parameter for the internal defence system. Two main fractions of E–S products were produced by T. ocellata, a low molecular fraction (low MW) and a high molecular fraction (high MW), weighing approximately 2 kDa and 40 kDa, respectively. Stage-dependent release of E–S products by transforming miracidia and mother sporocysts was indicated by the difference in proportions of these two fractions, which varied over time. After a culture time of 0–33 h, the smaller fraction was prominent whilst E–S products released between 33 and 72 h consisted of a higher proportion of the high MW fraction. After a culture of 72–96 h, lower and comparable quantities of both fractions were produced by the same batch of cultured parasites. Assaying the 2 fractions released during 0–33 h revealed that the high MW fraction had a suppressing effect on the bacterial clearance activity of haemocytes from juvenile snails and the low MW fraction activated haemocyte activity. Combined fractions obtained during 0–33 h had an activating effect on haemocyte activity but fractions separated from media collected at 33–72 h culture had a suppressing effect on the bacterial clearance activity of the haemocytes. The combined E–S fractions from the 72–96 h incubation had no effect on haemocyte activity. The present study shows that T. ocellata modulates the internal defence system of L. stagnalis by actively interfering with haemocyte function. MALDI-MS analysis of the low MW fraction revealed several components ranging from 450 to 1600 Da. Although it is as yet not clear which factor is responsible for the activating effect it is supposedly a peptide as it is pronase-sensitive and resistant to TFA and acetonitrile.

The physiological effects of synthetic replicates of the nematode FaRPs, AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), PF1 (SDPNFLRFamide), PF2 (SADPNFLRFamide), AF8/PF3 (KSAYMRFamide) and PF4 (KPNFIRFamide) were examined on muscle preparations of the liver fluke, Fasciola hepatica. Changes in contractility following the addition of the test compound were recorded using a photo-optic transducer system. Unlike the varied effects these peptides have on nematode somatic musculature, all were found to induce excitatory responses in the muscle activity of F. hepatica. While qualitative effects of the nematode peptides were similar in that they induced increases in both the amplitude and frequency of F. hepatica muscle contractions, they varied considerably in the potency of their excitatory effects. The threshold activity for each peptide was as follows: 10 μM, PF1 and PF2; 3 μM, AF1 and PF3; 1 μM, AF2; and 30 nM, PF4. The results demonstrate, for the first time, the cross-phyla activity of nematode neuropeptides on the neuromuscular activity of a trematode.

We present a mathematical model which is used to interpret the dynamics of the immunological response of a mouse host to infection with the filarial worm Onchocerca lienalis. The model mimics changes in worm burden over time post-infection and after reinfection and its behaviour provides a good description of experimental results. Measured production of T-cells and eosinophils is also compared with the predictions of the model. Our results show that the immune response mechanism proposed on the basis of experimental results, involving CD4+ T-cells and eosinophil destruction of the parasite, is supported by the insights gained from the mathematical model. Also, using the parameters estimated to describe the primary infection dynamics, the degree of acquired immunity to secondary infection is also well described by the model. Our analysis highlights the importance of obtaining quantitative measures of the many rate parameters involved in even the simplest interpretations of immunological responses to parasitic infection.

In earlier studies on the supposedly anaerobic metabolism of Leishmania promastigotes it was suggested that the reduction of fumarate to succinate functions as the main electron sink during anoxia. Interestingly, however, our preliminary results demonstrated that rhodoquinone, an essential component for efficient fumarate reduction in eukaryotes, was absent in L. infantum promastigotes. Therefore, we re-investigated the energy metabolism and succinate production of these promastigotes. Our studies demonstrated that L. infantum promastigotes could, to a certain extent, survive periods without respiration but had a low capacity for anaerobic metabolism. When oxygen could not be used as terminal electron acceptor, the degradation of glucose was severely inhibited, forcing the parasite to reduce its energy expenditure, which resulted in inhibited motility and proliferation. In addition, we studied the mechanism of succinate production under aerobic conditions and showed that in L. infantum promastigotes this succinate was mainly produced via an oxidative pathway, the Krebs cycle, and not significantly via fumarate reduction, which correlated with the absence of rhodoquinone. Taken collectively our studies show that L. infantum promastigotes depend mainly on respiratory chain activity for energy generation, have a poor capacity for anaerobic functioning, and go into metabolic arrest during anoxic conditions.

To study the infection dynamics of metacercariae of the digenean Cryptocotyle lingua, wild living Atlantic cod, Gadus morhua, were caged for 18 months close to the shore. Here they were exposed to naturally occurring transmission stages of the parasite. First, both the abundance and the variance to mean ratio of metacercariae increased, but during the second half of the study the abundance levelled out, and the variance to mean ratio showed a significant decrease. Host mortality was negligible throughout the study. Based on the relationship between pigment spots and metacercariae observed by skin digestion, there was no indication of density-dependent parasite mortality. We conclude that the infrapopulations of metacercariae on the caged cod probably were regulated by density-dependent host responses acting against the cercariae.

We have used nuclear (ribosomal ITS1) and mitochondrial (ND1) sequences to characterize human and pig isolates of Echinococcus granulosus collected by fine needle aspiration biopsy (FNAB) in Poland. The data indicate clearly that the Polish patients were not infected with the common sheep strain (G1 genotype) of E. granulosus, normally associated with human cystic hydatid infection. Instead, the hydatid parasite infecting the Polish patients shares very similar ND1 sequence with the previously characterized pig (G7) genotype but it also exhibits some clear differences. In particular, E. granulosus DNA from the Polish patients amplified a single ITS1 fragment in PCR and distinct ITS1–RFLP patterns were obtained after restriction digestion. The form of hydatid isolated from the Polish patients appears, therefore, to represent a distinct, previously undescribed genotype (designated G9) of E. granulosus.

A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine.

Adult schistosomes can be transferred surgically from donor C57BL/6 mice to the portal veins of naive recipients with complete success. This procedure bypasses larval development and the antibody response of the host is directed against, and can be used to identify, those antigens released only by viable, mature parasites. Serum collected from the recipient mice (WTS) was used in Western blotting studies to probe fractionated parasite protein. Twelve immunodominant proteins were identified, ranging in molecular weight from 14 to 208 kDa. The magnitude of the IgG response against each antigen could be divided into 2 categories, on the basis of optical densitometry of the blots. In addition, defined parasite fractions were probed with WTS by Western blotting, in order to determine the relative abundance and distribution of each antigen in schistosome tissue. To confirm and expand on these initial observations, oligospecific polyclonal antibody for each immunogen was affinity purified from Western blots; it was then used in immunocytochemistry to identify the sources of secretion for 8 of the 12 antigens, at the cellular level. From the results, it appeared that after the transfer of adult worms, the first antibodies detected were mostly directed against the gastrodermis. At later times additional reactivity was expressed against the tegumental membrane. These differences probably reflect the relative abundances of the gut and tegumental secretory products.

Antibodies to the core region of endotoxin (endotoxin core antibodies, EndoCAb), which cross-react with endotoxin from a range of Gram-negative bacteria, are maintained in relative homeostasis in health, but undergo marked changes in a number of different diseases associated directly or indirectly with endotoxaemic or septicaemic states. The levels of EndoCAb IgG in the blood and cerebrospinal fluid (CSF) of 35 late-stage sleeping sickness patients and 9 control individuals were measured by ELISA. EndoCAb levels were significantly elevated in the patient blood (mean EndoCAb value 290 MU/ml cf. control 182 MU/ml, P<0·001), and CSF (mean EndoCAb value 254 MU/ml cf. control 150 MU/ml, P<0·001). EndoCAb IgG levels correlated with endotoxin levels in patient blood (r=0·78, P<0·001), but not in the CSF and were not reduced 6 weeks following chemotherapy, unlike the endotoxin levels. It is concluded that late-stage sleeping sickness is associated with chronic exposure to endotoxins from Gram-negative bacteria.