Nucleotide sequences were obtained for the second internal transcribed spacer of the ribosomal gene repeat and for part of the mitochondrial cytochrome c oxidase subunit I gene from geographical isolates of Paragonimus westermani from Japan, China, Korea, Taiwan, the Philippines, peninsular Malaysia and Thailand. Sequences were obtained from several other species of Paragonimus for comparative purposes. Two groups were recognized within P. westermani: an NE group (China, Japan, Korea, Taiwan) which was relatively uniform and included both diploid and triploid forms, and a southern group (Malaysia, Thailand, Philippines), members of which were genetically distant from one another. According to both ITS2 and COI data, genetic distances among P. westermani isolates equalled or exceeded those between some distinct species of Paragonimus. The ITS2 sequences were conserved relative to COI sequences. Substitutions among the latter may be approaching saturation within the genus Paragonimus.

Infection with Toxocara canis is a common world-wide human helminthiasis, which rarely elicits central nervous system (CNS) impairment. A case-control study to investigate this discrepancy was carried out, in which the cases were 27 adult neurological inpatients for whom a definite aetiological diagnosis was lacking, and for whom positive immunodiagnosis of toxocariasis had been obtained, both in cerebrospinal fluid (CSF) and in serum. Two control groups were used. Controls were adult inpatients with other neurological diseases who had no evidence of T. canis infection of the CNS. Multivariate logistic regression analysis did not reveal any positive relation between case status and clinical signs. A significant association was observed between case status and an elevated CSF cell count. Rural residence, ownership of dogs, and dementia were shown to be risk factors for toxocaral infection of CNS. These results suggest that migration of T. canis larvae in the human brain does not frequently induce a recognizable neurological syndrome but is correlated with the association of several risk factors including exposure to dogs, a status possibly responsible for repeated low-dose infections.

Upon infection with a pathogen, eukaryotic cells can undergo programmed cell death as an ultimate response. Therefore, modulation of apoptosis is often a prerequisite to establish a host-pathogen relationship. Some pathogens kill macrophages by inducing apoptosis and thus overcome the microbicidal arsenal of the phagocyte. Apoptotic macrophages, on the other hand, can elicit an inflammation by secretion of proinflammatory cytokines. Shigella flexneri, the aetiological agent of bacillary dysentery, induces apoptosis in macrophages which, in agony, specifically release mature interleukin-1β (IL-1β). This cytokine attracts neutrophils (PMN) to the site of infection resulting in the massive colonic inflammation characteristic of bacillary dysentery. Shigellosis represents a paradigm of a proinflammatory apoptosis in a bacterial infection. The molecular link between apoptosis and inflammation is interleukin-1β converting enzyme (ICE) which is activated during macrophage apoptosis and binds to IpaB, a secreted Shigella protein.

A trickle infection experiment was undertaken to study in detail the population dynamics of Oesophagostomum dentatum in pigs. Three groups of 32 pigs were inoculated via the feed twice weekly with 100 (Group A), 1000 (Group B) or 10000 (Group C) O. dentatum infective larvae (L3). Five pigs from each group were killed 2, 4, 8, 12, 16, and 20 weeks after the first inoculation (p.i.) to determine their worm burdens. Weekly faecal egg counts were determined. At slaughter, worms were counted, differentiated according to sex and developmental stage, and their length measured. Faecal egg counts ranked with dose rate until week 15, but later were more variable. The proportion of the total number of L3 administered which were recovered at slaughter inversely ranked with dose rate. In group C it decreased over time, whereas in groups A and B there was no consistent pattern. Worm fecundities (epg/female) in groups A and B were higher than in group C. The lengths of the female worms increased over time, whereas the lengths of the male worms remained approximately constant from week 8. The study suggests reduced establishment of incoming larvae and lower fecundity of the female worms at high dose levels.

Hosts exert selection pressures on their parasites and it is often assumed that host–parasite coevolution with each host is less intense in a generalist parasite than for a parasite with a narrow host range. Selection pressure on the parasite, however, is rather determined by host specificity, i.e. the relative importance of each host, than simply by the range of hosts. The determination of host specificity requires an assessment of the prevalence and intensity of parasite infestation within each host's nests, as well as the local abundance of each host species. Since the hen flea, Ceratophyllus gallinae, is a rather generalist parasite of birds it could be concluded that there has been weak coevolution with each of its hosts. By reviewing the literature on the prevalence and intensity of hen flea infestations in bird nests we estimated the number of individuals produced in the nest of each host species. The comparative analysis shows (1) that the prevalence of infestation is highest in hole-nesting avian families, (2) that prevalence and intensity of infestation among bird families are highly correlated, and (3) that hole-nesting Paridae have the highest intensities of infestation and harbour the majority of the flea population. These results underline the fleas' potential for coevolution with Paridae despite their extensive host range.

Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (α, β, γ and δ), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoform patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.

Several monoclonal antibodies (Mabs) were raised against the L1 muscle stage (L1M) of Trichinella spiralis (Ts) and Trichinella pseudospiralis (Tp). Western blot analysis of various antigenic preparations established that Mabs described by different authors recognized 8 antigenic fractions (TSL1–TSL8) in crude extracts of infective larvae. The TSL1 fraction was immunodominant and present on the cuticle of different parasite stages. Mabs against Trichinella T5 (T5) and Ts were selected in order to extend the previous studies to another Trichinella phenotype. Only 35% of the selected Mabs recognized linear epitopes and 71% reacted with soluble or excretory–secretory antigens in a dot blot procedure and ELISA test. The targets of the Mabs were identified by immunoprecipitation with [35S]methionine-labelled L1M worm lysate. Mabs prepared from mice immunized with the whole parasite (T5) recognized a wider panel of antigens in different parasitic organs. Seven antigenic structures were distinguished on the cuticle and several epitopes were identified in the gut, haemolymph and stichocytes. Eleven antigenic groups were established according to their indirect immunofluorescence pattern on cross-sections of the worm. Monoclonal antibodies raised against Ts soluble antigen mainly recognized epitopes in stichocytes and on the cuticle surface. All the selected Mabs recognized T5 and Trichinella britovi (Tb) strengthening the link between these 2 species. Four Mabs were used to differentiate antigenic structures among 6 Trichinella phenotypes and to develop a new tool to follow gene flow within the Trichinella genus.

The influence of nutrient level, temperature and larval density on the trapping of Heligmosomoides polygyrus L3 by the nematophagous fungi Arthrobotrys oligospora, Duddingtonia flagrans and Monacrosporium megalosporum were investigated by quantification of trapped nematodes. All 3 factors were found to have a significant effect on the number of larvae trapped by A. oligospora and M. megalosporum. Decreased nutrient concentrations resulted in increased trapping for these 2 fungi, but nutrient availability was not found to have a significant effect on trapping by D. flagrans. The 3 fungi were found to have similar responses to temperature, with peak trapping occurring at or near the optimum growth temperatures. Nematode trapping was found to be density dependent for all 3 fungi, with increased percentage trapping at increased larval densities. Comparison in a single experiment of the relative importance of these factors to each fungus showed that nutrient level was the main factor influencing trapping by A. oligospora, whereas D. flagrans and M. megalosporum were more dependent on larval density.

Different populations of the whitespotted rabbitfish, Siganus sutor, were examined for ectoparasites: adults from the Mombasa area (sampled in December 1990) and different age classes (adult, subadult and juveniles) from Gazi Bay (sampled in December 1992 and August 1993). The most common gill parasites were: the monogeneans Pseudohaliotrema sp., Tetrancistrum sigani and Microcotyle mouwoi, the copepods Hatschekia sp., Pseudolepeophtheirus sp. and juvenile Caligidae, and prazina larvae of the isopod Gnathia sp. Adult siganids had a higher parasite load than subadults. Juvenile rabbitfish did not harbour any gill parasites. Temporal differences in the parasite load of subadult rabbitfish were observed for M. mouwoi (highest in the December samples) and for juvenile Caligidae (highest in August). The microhabitat of the 5 most common gill parasites was species specific. Most parasite species showed distinct site preferences with respect to both gill arches and gill sectors, within the gill arches. Niche breadth of the different gill parasite species was independent of the abundance of any of the other species present. However, niche breadths of M. mouwoi, Tetrancistrum sp. and Hatschekia sp. increased with their own abundance. This suggests that interspecific competition for space is low and that intraspecific factors could play an important role in the microhabitat choice of these gill parasites. The hypothesis that niche restriction leads to higher intraspecific contact and an enhancement of chances to mate was tested on 2 monogenean species, Pseudohaliotrema sp. and Tetrancistrum sigani. Their highly aggregated distribution over the gill filaments, leading to increased intraspecific contact, is consistent with the hypothesis.

In order to replicate efficiently within the host, viruses have evolved multiple strategies to evade the host's immune system. In many cases viruses have actually hijacked various components of the host's immune system to ensure their own survival. One such strategy is the expression of virus encoded cytokines and cytokine receptors. Members of the poxvirus and herpesvirus families have been particularly successful with this strategy. The study of virus survival strategies provides important information regarding both virus biology as well as information about the immune system itself.

The anthelmintic ivermectin has a number of effects on nematodes which result in changes in behaviour, particularly locomotion, including paralysis and an inhibition of feeding. This paper describes the application of an in vitro pharmacological approach to further delineate the action of ivermectin on feeding behaviour. Contraction of Ascaris suum pharyngeal muscle was monitored using a modified pressure transducer system which detects changes in intrapharyngeal pressure and therefore contraction of the radial muscle of the pharynx. The pharynx did not contract spontaneously. However, serotonin (5-HT, 100 μm) stimulated rhythmic contractions and relaxations (pumping) at a frequency of 0·5 Hz. γ-Aminobutyric acid (GABA) and glutamic acid inhibited the pumping elicited by 5-HT. The duration of inhibition was concentration dependent (1–1000 μm) with a threshold of 1 μm and 10 μm respectively (n=8). Ivermectin also inhibited pharyngeal pumping (1–1000 nM). At lower concentrations, ivermectin (1–10 pM) potentiated the GABA and glutamate inhibition, so that inhibition occurred at concentrations which were below threshold in the absence of ivermectin. These data provide evidence that the pharynx is a site for the action of ivermectin. Thus interruption of pharyngeal processes such as, feeding, regulation of hydrostatic pressure and secretion may provide a new site of anthelmintic action.

A single form of cholinesterase was detected in the parasitic nematode Parascaris equorum and purified from a low-salt Triton X-100 extract of whole animals by affinity chromatography on an edrophonium–Sepharose matrix. Based on gel-filtration chromatography, sedimentation analysis and SDS–PAGE, such a cholinesterase is an amphiphilic globular (G2) dimer (125–129 kDa, 6·1 S). It includes some hydrophobic domain other than phosphatidylinositol, which gives auto-aggregation, detergent interaction and also anchors the molecule to the cell membrane. The enzyme, probably functional in cholinergic neurotransmission, is an acetylcholinesterase showing a fairly low substrate specificity with thiocholine esters. Electrostatic interactions seem to play a major role in the catalytic activity. Studies with inhibitors gave complete inhibition with 1 mM eserine, low sensitivity for procainamide and for tetra(monoisopropyl)pyrophosphortetramide as well as higher inhibition with edrophonium chloride and 1,5-bis(4allyldimethylammoniumphenyl)-pentan-3-one dibromide. The enzyme also showed excess-substrate inhibition with acetylthiocholine. No cross-hybridization occurred between the gene(s) encoding acetylcholinesterase in P. equorum and ace-1 from the free-living nematode Caenorhabditis elegans. The expression of a single cholinesterase form in P. equorum, unusual in free-living nematodes, could be due to parasitic life adaptation with resulting reduction of locomotor activity.

AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) are endogenous RFamide-like peptides isolated from the parasitic nematode Ascaris suum. Here the actions of these peptides on the somatic musculature of Ascaris have been investigated and compared to the action of acetylcholine (ACh), the excitatory transmitter at the neuromuscular junction. ACh, AF3 and AF4 contracted muscle with EC50s of 13±1 μM, 24±6 nM and 37±2 nM, respectively (n=6). The muscle cells were depolarized by ACh (3 μM; 5·2±0·4 mV, n=42), AF3 (1 μM; 2·6±0·3 mV, n=19) and AF4 (1 μM; 3·3±0·4 mV, n=19). EC50s were 681±329 nM (AF3) and 901±229 nM (AF4), but an estimate could not be made for ACh due to muscle contraction at concentrations greater than 10 μM. The depolarization to 3 μM ACh was abolished by the nicotinic receptor antagonist mecamylamine (10 μM; n=5) but the responses to the peptides were not (111±7% and 108±17% with respect to control; n=5). The depolarization elicited by ACh was reduced to a greater extent by a 50% reduction in extracellular Na+ concentration than the response to AF3 and AF4 (P<0·02). Cobalt was more effective at blocking the AF3 and AF4 depolarizations than those to ACh. These observations suggest that AF3 and AF4 contract Ascaris muscle without an action at the Ascaris nicotinic receptor. Furthermore, the ionic mechanism through which AF3 and AF4 depolarize Ascaris muscle is different from that for ACh. ACh, AF3 and AF4 were also found to contract Ascaridia galli somatic muscle with EC50s of 13±3 μM, 721±236 nM and 371±177 nM, respectively (n=7). The muscle cells were depolarized by ACh (EC50=14±5 μM, n=5), AF3 (EC50=5±3 μM, n=4) and AF4 (EC50=10±5 μM, n=4). Therefore the response to these peptides is not unique to Ascaris and they may subserve a functional role in the motor nervous system of parasitic nematodes.

Chronic infection is a prominent feature of many intestinal nematode infections in man and animals. It is also clear that in such situations host immunity is activated but is unable to induce a protective response. A great deal of work has shown that genetic control of host immunity contributes to the variation in worm burdens often observed in the field. There is increasing appreciation, however, of the capability of infectious agents themselves to modulate the host immune response and potentiate their own survival. Using an immunologically well defined model of intestinal nematode infection in mice (Trichuris muris) we have shown that parasite derived molecules share cross reactive epitopes with the host cytokine interferon-γ using cytokine specific monoclonal antibodies in ELISA, Western blotting and immunoprecipitation assays. Furthermore, the parasite molecules can be shown to bind to the interferon-γ receptor and induce change in lymphoid cells similar to those induced by murine interferon-γ. The functional activity of the molecule in vivo remains to be determined. Previous studies have established that interferon-γ is critical for progression to chronic T. muris infection in mice and, therefore, it raises the distinct possibility that the production of an interferon-γ homologue by the worm may be one mechanism whereby the parasite is able to interfere with the regulation of the host immune response and potentiate its own survival.

Electrophoresis of secretions collected from Globodera pallida revealed a smeared region between 25 and 50 kDa, and a single band of <20 kDa. The secretions were used to raise an antiserum (LW1). Immunoblotting of parasite homogenates with LW1 differentiated G. pallida from its sibling species G. rostochiensis and revealed differences between different populations of G. pallida and G. rostochiensis. Indirect immunofluorescence studies with LW1 indicated that at least some of the secretions were surface localized and that antibody binding to the nematode surface was periodate sensitive. Periodate sensitivity indicated that these differences could be due to glycosylation differences. Glycosylation differences were also detected by blotting nematode homogenates with the lectin wheat germ agglutinin (WGA). WGA was also able to differentiate between G. rostochiensis which gave 2 bands at 130 kDa and 110 kDa, and G. pallida which produced 2 bands present at 120 kDa and 110 kDa. Further localization studies using immunoelectron microscopy demonstrated that antibody binding could be seen to secretions found in the pump chamber of the metacorpal bulb at the base of the stylet. From further specimens it could be observed that the contents of the subventral glands were heavily labelled, indicating that the material seen in the metacorpal bulb had originated from the subventral glands.

Electrophysiological techniques and behavioural assays were used to determine the response of males of Globodera rostochiensis to 4 fractions of the homospecific sex pheromone from virgin females, separated using reverse-phase high-pressure liquid chromatography. Sex pheromone activity was confined to 2 of the fractions. The sex pheromone of G. rostochiensis appears to be composed of several weakly basic, polar compounds.

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) ([ges ]40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3% routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin–Cohn's fraction V) promoted the growth of B. divergens with high PPE (>30%) close to those obtained in RPMI–10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

In an experiment to study the infectivity, growth and virulence of Schistocephalus solidus in their first intermediate host, copepods of the species Macrocyclops albidus were kept singly and exposed to up to 9 coracidia. Eleven or 14 days post-infection (p.i.) the presence and growth of the cestode larvae relative to survival, growth and reproduction of their host was determined. As expected, the probability of a copepod becoming infected increased with increasing numbers of parasites administered. However, the chances of a single coracidium establishing in a copepod also increased with increasing numbers of coracidia administered, which indicates that the parasites profit from a dilution effect of the host's defence. Copepod size or developmental stage had no significant effect on the infection, but 14 days p.i., constraining effects of copepod size on the growth of the parasites were apparent. Moreover, procercoids in multiple infections grew smaller and developed their cercomer at a smaller size than those in single infections. No significant effect of the parasite on host mortality was found within the observation period. However, growth between the 5th copepodid stage and adult stage was negatively affected by infection. An infection with S. solidus was also strongly linked with host reproduction: infected females were more likely to bear an egg sac at the end of the experiment than non-infected ones. These egg sacs, however, contained fewer eggs.

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.

Schistosome parasites, despite being multicellular organisms several millimetres in length, can survive in the bloodstream of mammalian hosts for decades. The remarkable and complex adaptation exemplified in the host–parasite relationship in schistosomiasis may include not only immune evasion by the parasite, but also immune exploitation. While the developmental and adult stages of the parasite are by and large invisible to the immune response, the parasite egg induces a granulomatous reaction which not only protects the host from a diffusible parasite toxin, but also is required for normal transmission of parasite eggs from the host to the external environment. Other possible mechanisms of immune exploitation by schistosomes are discussed including skewing of cytokine responses, effects of cytokines on worm fecundity, exploitation of endothelial cell adherence, and induction of IgE.