We used the PCR–SSCP method followed by sequencing in order to assess the genetic variability of coding and non- coding parts of the genome of Echinococcus granulosus (Cestoda; Taeniidae) and to test whether or not the parasite populations are mainly self-fertilizing. For this, we analysed a sample of 110 E. granulosus metacestode isolates collected from different geographical regions (Southern Brazil, Europe and Australia) and from different intermediate hosts (ovine, bovine, human, macropod, swine and equine). Using appropriate controls, we were able to identify 4 strains in that sample (sheep, cattle, pig and horse strains). The high degree of genetic differentiation between strains, but not within, and the monomorphism found in most loci (EgAg4, EgActII, EgHbx2 and EgAg6 – non-coding – EgAgB/1 and EgND1 – coding) indicated that they are largely selfed. On the other hand, outcrossing was also shown to occur, since 5 potential hybrid genotypes between cattle and sheep strains were found in populations of Southern Brazil, but absent in other geographical areas. We suggest that both processes are adaptive. The article also reports, for the first time, the occurrence of the E. granulosus cattle strain in South America.

The surface coat of the infective larvae of the parasitic nematode Trichinella spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surface probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron microscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity of PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4°C, suggesting the shedding process required metabolic energy. Pre-labelled, unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated failed to re-label with the probe, suggesting that activation provides an irreversible trigger for surface changes. PKH26, therefore, is a useful marker for larval activation. Examination of the shed coat material by scanning electron microscopy revealed 2 types of morphologies; one comprising thin multilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat material demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treatment and a 47–60 kDa set of protein(s). Analysis of the surface lipids by electrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14[ratio ]2) and an unidentifiable component of 339·4 Da. These two lipids constituted 36·9% and 36% by mass of surface coat lipids respectively. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48[ratio ]4), PE (C40[ratio ]4 and C38[ratio ]4), PS (C40[ratio ]4), lysoPC (C20[ratio ]2 and C18[ratio ]2) and lysoPA (C14[ratio ]2). These observations are discussed with respect to the role of the surface coat and its shedding in the T. spiralis host–parasite relationship.

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.

Onchocerciasis (‘River Blindness’), caused by the filarial nematode Onchocerca volvulus is of major public health importance in West Africa. Ivermectin, a drug originally developed for veterinary use, is now being incorporated in control strategies but whilst it has potent efficacy against L1 larvae (microfilariae), ivermectin is not lethal to adult (L5) O. volvulus, nor to adults of the related cattle parasite O. ochengi. We have exploited this model to determine if ivermectin has prophylactic activity against naturally transmitted, O. ochengi infections in a controlled, prospective study in northern Cameroon. Calves were treated monthly with ivermectin at either 200 μg/kg or 500 μg/kg for 21 months. None of 15 treated calves developed adult worm infection, whereas 5/6 untreated controls became infected (P<0·001) with a total of 54 O. ochengi nodules, and all 5 developed patent microfilaridermia. These results have significant implications for the use of ivermectin in humans, and suggest that strategic chemotherapy at times of maximal transmission will confer prophylactic as well as therapeutic benefits.

In this study, we employed a mutation scanning approach for the direct visual display of genetic variability in mitochondrial DNA (mtDNA) fragments within and among populations of Schistosoma japonicum from the People's Republic of China. Fragments of the NADH dehydrogenase 1 gene (ND1) and the cytochrome c oxidase subunit I (COI) were individually amplified from parasite DNA by polymerase chain reaction (PCR), denatured and subjected to single-strand conformation polymorphism (SSCP) analysis. Using ND1 and COI fragments, individuals representing different genotypes could be readily identified based on characteristic and reproducible SSCP profiles. The results demonstrated the usefulness of SSCP for the direct visual display of low-level sequence variation in mtDNA of S. japonicum prior to DNA sequence analysis. This approach has important implications for studying the genetic structure and biology of S. japonicum populations, and for analysing the inheritance of mitochondrial DNA.

Polymerase chain reaction–ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR–RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the camel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.

Vaccination of lambs with the membrane-bound (S3) thiol-Sepharose binding protein (TSBP) fraction derived from the gut of Haemonchus contortus confers significant protection against homologous challenge. The S3 TSBP peptide profile is dominated by a major protein of ca. 60 kDa which is strongly recognized by antisera from sheep demonstrably protected following immunization with S3 TSBP. In an attempt to identify this protein, sera from protected lambs were employed to screen a λgt11 cDNA library of the adult parasite and resulted in the isolation of numerous clones encoding a homologue of the mitochondrial enzyme, glutamate dehydrogenase (GDH). GDH enzyme activity was readily demonstrable in S3 TSBP material and immunolocalization studies showed that the enzyme was localized to the cytoplasm of the parasite's gut. Furthermore, the enzyme appeared to be developmentally regulated, with both GDH mRNA and protein expressed almost exclusively during the blood-feeding parasitic stages.

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.

This study characterized antigens of Trichinella spiralis and T. pseudospiralis muscle larvae recognized by mice infected with the worms. Two-dimensional (2-D) Western blot analysis revealed some profile of antigenic peptides including: (1) molecular weight (MW); (2) isoelectric points (pI), (3) reactivity to well-defined monoclonal antibodies (mAb) and (4) cross-reactivity between the 2 species. Antigenic peptides of T. spiralis consisted of about 100 spots. The MW ranged from 22 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. spiralis excretory–secretory (E–S) peptides migrating at 40, 45 and 50 kDa, and the mAb against TSL-4 stained non-E–S peptides. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots. The MW ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. pseudospiralis E–S peptides migrating at 35 and 45 kDa, and the mAb against TSL-4 stained non-E–S peptides. Two-dimensional Western blots showed that the E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other. The non-E–S peptides were, however, not recognized by T. pseudospiralis-infected sera but were recognized by T. spiralis-infected sera. An immunoelectron microscopical study showed the similar result that stichocyte granules and cuticle surface (known to contain E–S antigen) had cross-reactive antigens between the two species. T. pseudospiralis-infected sera stained very weakly the cuticle inner layers and haemolymph (known to contain non-E–S antigen). This evidence implies that mice infected with T. pseudospiralis do not evoke antibodies against non-E–S antigen at the detectable level.

A recombinant cysteine protease inhibitor, onchocystatin of the parasitic nematode Onchocerca volvulus, was tested for its role in microfilarial development in the simuliid vector. Onchocystatin was found to be present in female adults and skin microfilariae of the bovine parasite O. ochengi, the closest relative of O. volvulus. In addition the inhibitor could be detected as an excretory–secretory (E–S) product of the microfilariae. Co-injection of onchocystatin and the O. ochengi microfilariae into the surrogate vector Simulium ornatum s.l. significantly enhanced the recovery rates of the parasite within 24 h into the infection (P>0·001). The findings suggest a possible role of onchocystatin in the evasion by the parasite of the immune response of its vector.

Both CEF6, a cation-exchange fraction of soluble Schistosoma mansoni egg antigens (SEA), composed of the 2 antigens, alpha-1 and omega-1, and haemocyanin from the keyhole limpet, Megathura crenulata, have shown potential for immunodiagnosis of human schistosomiasis. Possible cross-reactivity between antigens in SEA and keyhole limpet haemocyanin (KLH) was explored by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with KLH, SEA, CEF6, alpha-1, omega-1, or egg antigen k5. Both immunoassays revealed a high degree of serological cross-reactivity between the schistosome egg antigens and KLH, much of it due to sodium periodate- sensitive epitopes. Cross-reactivity with schistosome antigens with proven diagnostic efficacy may thus, in part, explain the usefulness of KLH for the diagnosis of human schistosomiasis mansoni.

To examine levels of heat shock proteins (HSPs) in host responses to helminth infection, rats were infected with Trichinella spiralis. The kinetics of HSP25, HSP60, HSP70 and HSP90 production in the liver and muscle of infected rats was compared with that of non-infected controls. HSPs were detected using electrophoretic analysis of fixed amounts of proteins (0·02 mg) and then blotting and incubation of membranes with polyclonal anti-HSP25 antibody, or monoclonal antibody against HSP60, or HSP70 or HSP90. Quantitation of blotted separated polypeptides reactive with the specific anti-HSP antibodies was achieved using an image analyser. Enhancement of HSP25 production was observed in the liver of infected rats, whereas muscle from the same rats exhibited enhanced production of HSP25 and HSP60 one day after infection only. These data indicate that HSPs levels can be used successfully to measure stress injury brought about by helminth infection in organs and tissues of the host.

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.

The subsets of lymphocytes and cytokines regulating the site-specific immune response in experimental cysticercosis (Taenia crassiceps) are not known. This study investigated the cells present at the site of infection (PECs) using flow cytometry and measured the cytokines produced by these cells through 50 days of infection. The results showed an expansion of B220+CD5+, B220+CD5−, αβTCR+CD4+ and CD8+ cells coincident with a transient increase in IL-10 production. After the initial increase, the percentage of B220+ CD5− and helper T cells decreased with a concomitant decrease in IL-10 production. CD8+ T cells continued to increase throughout infection and γδTCR+ cells increased after 10 days of infection. PECs demonstrated an increased IFN-γ and IL-4 production throughout infection when stimulated with larval antigens. Because a Th2-type polarization has been shown for spleen cells from infected BALB/c mice, cytokine profiles of spleen cells and PECs in response to ConA and larval antigens were compared. ConA and antigen-specific stimulation of spleen cells from 50-day-infected mice produced increased amounts of IL-10 while PECs showed a decreased IL-10 production suggesting that anatomically distinct lymphoid populations produce different cytokines and promote different types of responses. Surprisingly, late in infection the levels of IL-4 and IFN-γ in serum increased substantially (460-fold and 100-fold, respectively). The systemic immune response of BALB/c mice during experimental cysticercosis, therefore, is a mixed Th1/Th2-type response.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES–62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway – phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

The cellular inflammatory response in the small intestine of 21 goats infected with Schistosoma bovis was phenotypically characterized by immunohistochemistry between 6 and 32 weeks post-exposure, with particular reference to perioval granulomatous reactions. Macrophages of granulomas consistently expressed MHC class II molecules, whereas multi-nucleated giant cells in general did not. Most granulomas contained moderate infiltrates of CD2+ (CD4+ or CD8+) and γ/δ (T19+) T cells, whereas B lymphocytes were sparse. Intact extravascular mucosal eggs, lacking appreciable cellular reactivity on plain histopathology, displayed surrounding collars of MHC class II+ macrophages. Gamma/delta T cells and MHC class II+ macrophages were the predominant cell types in perivascular inflammatory cell clusters in the submucosa. The phenotypic cellular composition of granulomas did not change appreciably with duration of infection. The results indicate the importance of MHC class II-restricted immune events in the caprine S. bovis egg granulomas and also suggest a role of γ/δ T cells in their pathogenesis. It is hypothesized that the early appearance of perioval macrophage collars may serve to protect eggs from ovicidal host defence mechanisms, facilitating excretion and continuation of the life-cycle.

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.

In the present study, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was developed to identify 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory–secretory (E–S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the HinfI or DdeI endonuclease restriction of the 2800 bp fragment; T. nativa by the RsaI restriction of the 2800 bp fragment, or by the AluI restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the AluI restriction of the 1250 bp fragments, and can be discriminated between them by the SspI restriction of the 2800 bp fragment; T. pseudospiralis by the MspI restriction of the 372 bp fragment; T. nelsoni by the HhaI or AluI restriction of the 2800 bp fragment; Trichinella T5 by the HhaI restriction of the 2800 bp fragment; Trichinella T6 by the AluI restriction of the 1250 bp fragment; and Trichinella T8 by the SspI or RsaI restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi, Trichinella T5 and T6.

The humoral antibody response and the number of pronephric antibody-secreting cells were examined in naturally Bothriocephalus acheilognathi-infected carp. Cyprinus carpio, and in those injected intraperitoneally with an extract of the cestode. In the extract-injected fish, specific antibody was detected 3 weeks after a second injection given 2 weeks after the primary injection, and antibody levels persisted for more than 200 days. A third injection also enhanced the antibody level in the extract-injected carp. The numbers of antibody-secreting cells were significantly higher in carp injected 3 times with the extract than in the control. In naturally-infected fish, the serum antibody levels and the number of pronephric antibody-secreting cells were higher in infected fish than in uninfected individuals although this difference was not statistically significant. The relevance of these results to immune protection against infection is discussed.

In a fishing community on Lake Albert in Uganda the pattern of intensity of Schistosoma mansoni infection 6 months after treatment with praziquantel was found to be very similar to reinfection patterns seen in previously studied endemic communities: the profile peaks sharply at around the age of 10 years falling away rapidly to much lower levels in adults. This is in stark contrast to the patterns of water contact, which differ greatly between fishing and non-fishing communities. On Lake Albert, adults appear to be more heavily exposed than children. From these observations we conclude that adults are physiologically (perhaps immunologically) more resistant to infection after treatment than children.