Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

Irreversible pathological lesions were noted in the organs of mice infected with 1 of 3 rodent malaria species: Plasmodium chabaudi chabaudi, P. vinckei petteri and P. yoelii nigeriensis at different times during the course of the primary parasitaemia and long after microscopical clearance of the parasites. Moreover, similar lesions were also obtained when parasite levels were kept below 1%by subcurative drug treatment. The frequency and severity of the lesions correlated with the duration of the infection. Accumulation of tissue damage during chronic low-grade malaria infections has implications for the design of control measures.

Proliferative kidney disease (PKD), a condition associated with high mortality in salmonid fish, represents an abnormal immune response to the presence of an enigmatic myxozoan, which has been designated simply as PKX organism because its generic and specific status are obscure. Phylogenetic analyses of partial sequences of the 18S rDNA of PKX and of myxozoan parasites infecting the bryozoans Cristatella mucedo, Pectinatella magnifica and Plumatella rugosa, including the previously named Tetracapsula bryozoides from C. mucedo, showed that these taxa represent a distinct clade that diverged early in the evolution of the Myxozoa before the radiation of the other known myxozoan genera. A common feature of the myxozoans in this clade may be the electron-dense sporoplasmosomes with a lucent bar-like structure, which occur in T. bryozoides and PKX but not in the myxozoans belonging to the established orders Bivalvulida and Multivalvulida. Variation of 0·5–1·1% was found among the PKX 18S rDNA sequences obtained from fish from North America and Europe. The 18S rDNA sequence for T. bryozoides showed that it is a distinct taxon, not closely related to PKX but some sequences from myxozoans infecting 2 of the bryozoan species were so similar to those of PKX as to be indistinguishable. Other sequences from the new myxozoans in bryozoans at first appeared distinct from PKX in a maximum likelihood tree but, when analysed further, were also found to be phylogenetically indistinguishable from PKX. We propose that at least some variants of these new myxozoans from bryozoans are able to infect and multiply in salmonid fish, in which they stimulate the immune reaction and cause PKD but are unable to form mature spores to complete their development.

Activity of the following glycosidases was detected in the plasma of the freshwater snail Biomphalaria glabrata: β-D- fucosidase, β-D-glucosidase, β-D-galactosidase, β-D-mannosidase, β-D-glucuronidase, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-glucosaminidase, and lysozyme. At the physiological pH (7·2–7·4) of snail haemolymph, enzymatic activity was about 10–50% of the maximum activity at each enzyme's respective acid pH-optimum. Schistosome-susceptible B. glabrata showed lower plasma protein concentration and significantly lower enzymatic activities (U/mg protein) than schistosome-resistant snails. Changes in glycosidase activity levels correlate with the progress of infection. After successful schistosome invasion, activities of plasma glycosidases but not the concentration of total plasma proteins increased significantly during the first 2 days in both snail strains. Thus, most tegumental glycoproteins of schistosome larvae can be altered by humoral host glycosidases. The detection of only very low activities of hexosaminidases leads to the hypothesis that GalNAc/GlcNAc may be involved in the process of non-self recognition. At 4 days post-infection, glycosidase activities were identical or slightly below the levels found in naive snails. At this time of infection the parasite is encapsulated and destroyed by haemocytes of resistant snails. In susceptible snails, however, the schistosomes have transformed into sporocysts and will complete their life-cycle without eliciting effective defence reactions. After >30 days post-infection, when cercariae are fully developed in susceptible snails, plasma protein concentration decreased significantly, whereas glycosidase activities were elevated.

The Schistosoma mansoni protein, SPRM1lc, is a light chain member of a new family of heterodimeric amino acid permeases. These proteins require covalent association with a type II glycoprotein (like h4F2hc) for functional surface localization when expressed in Xenopus oocytes. We previously reported that, when co-expressed with h4F2hc, the transport properties of SPRM1lc resemble system y and y+ while its human homologue, E16, functions as an L-type permease. Here we extend the functional characterization of SPRM1lc in oocytes and show by competitor studies that its amino acid transport capacity is similar to that of whole adult schistosomes. We demonstrate by Northern and Western analysis that SPRM1lc is expressed within both larval and adult schistosomes. In all stages, SPRM1lc is associated into a high molecular weight complex that can be disrupted by reducing agents, consistent with the hypothesis that a significant fraction of the endogenous SPRM1lc is linked by a disulphide bond to an uncharacterized schistosome amino acid transporter heavy chain. Immunofluorescence localization detects SPRM1lc in miracidia, daughter sporocysts and adult worms. Confocal microscopy demonstrates that SPRM1lc is found in the apical membrane of the syncytial, double-lipid bilayer tegument which surrounds adult worms. Aqueous biotinylation studies on living worms show that SPRM1lc is exposed on the host-interactive surface of this tegumental membrane. Host exposed, functionally important surface proteins such as SPRM1lc could form the basis of an effective schistosomiasis vaccine. These studies are the first to describe a helminth amino acid transporter, and the first to characterize an invertebrate heterodimeric amino acid transporter.

The development of nasal avian schistosomes of the genus Trichobilharzia in their final host is poorly known. Therefore, an experimental infection of ducklings (Anas platyrhynchos f. dom.) by T. regenti was performed. The infection resulted in leg paralysis and orientation/balance disorders of birds. The examination of the duck's spinal cord and brain confirmed the presence of developing parasites in pre-patent as well as patent periods. The absence of the worms in other tissues strongly supports our hypothesis that the parasite migrates through the central nervous system (CNS) to its final location in bird nasal mucosa. The injury level is probably dependent on number of parasites as well as yet unknown host factors. The affinity to the CNS seems to be high; also by exposure of experimental animals to low cercarial doses the growing worms in the CNS were found. In addition to the generally accepted view that bird schistosomes may cause cercarial dermatitis of mammals (including man), there is evidence of a partial development of T. regenti in mouse CNS; in certain cases leg paralysis was also recorded. Therefore, the pathogenesis spectrum caused by bird schistosomes in birds/mammals needs to be reconsidered.

The biodiversity of Trypanosoma cruzi is one of the main factors complicating the understanding of its molecular epidemiology. As an alternative to classical genetic methods, investigators have used DNA-mediated transformation techniques to study this diversity. Recently, transient expression data were shown to correlate with the genetic data. This led investigators to speculate on a potential speciation within clonal populations of Trypanosoma cruzi. To further test the phylogenetic significance of transient expression analysis in Trypanosoma cruzi, multiple plasmids were used to drive the expression of a reporter gene in different parasite populations. In our study, population specific expression of the reporter gene was observed but the variability revealed by these transient expression assays was very large and did not follow the grouping of Trypanosoma cruzi populations in 2 lineages. In this report, we discuss some of the limitations of transient assays on such a diverse parasite.

Haemoflagellates of the genus Trypanosoma are prevalent in freshwater fishes and are transmitted by leeches as vectors. As demonstrated by sequence comparisons of nuclear small subunit rRNA genes, trypanosomes isolated from several fish species at different localities can be divided into at least 2 closely related types, designated Type A and Type B. A clone derived from a Type A isolate from carp (Cyprinus carpio) was used to study the anti-parasite immune response in specified pathogen-free outbred carp. Infection leads to an initial rise in parasitaemia in the blood followed by a sharp decline in all fish (acute phase). Thereafter, in some carp, parasites become undetectable both in the blood and in internal organs while, in others, low numbers can be found in the blood for up to 1 year (chronic phase). Fish that have controlled an acute infection with the clone are not only protected against an homologous challenge infection, but also against the infection with parasite lines derived from carp in the chronic phase of infection. Passive immunization experiments with IgM purified from serum of recovered carp indicate that the infection is controlled by antibodies. The anti-parasite antibody level in recovered carp remains high for many months although the parasitaemia is controlled at very low levels and the half life of IgM, t1/2=22·5 days, is comparatively short. The effective control of trypanosomes in laboratory infections is in contrast to the high prevalence in natural and farmed freshwater fish populations.

The antibody responses of 8 cattle experimentally infected with Onchocerca ochengi to 18 recombinant O. volvulus antigens were measured by ELISA. In addition to establishing antigenic cross-reactivity between the species, the dynamics of antigen-specific responses were examined to assess how the recognition of the antigens compared to the known stage-specificity of expression. Six cattle responded to all of the antigens and 2 animals responded to all but 1. The dynamics of the recognition of 4 antigens (B20, MOv-2, MOv-14 and OvNHR2 02E1) were characterized by rapid seroconversion following infection. Antibody levels to 2 antigens (Ov7 and OvALT-1) increased gradually over the course of infection. Antibody levels to 4 antigens (OvTPX-2, OvL3Chitinase, Ov103 and Ov9m) reached maximum levels coincident with the onset of patency. The levels to 3 antigens (OvProalf C50, OvAldolase, Ov39) varied little over the course of infection. Responses to antigens with functional similarities (OvSOD1, OvSOD2 and OvSOD3 or OvGST1 and OvGST2) showed comparable temporal profiles. This study demonstrates the high degree of immunological cross-reactivity between the antigens of O. volvulus and O. ochengi. The immunogenicity of antigens varied over the course of infection in an antigen-specific manner, which not always reflected developmentally regulated expression of the corresponding gene, possibly owing to cross-reactive epitopes on distinct parasite products.

Detection of Onchocerca volvulus larvae in vector populations is of prime importance in the assessment of the effectiveness of onchocerciasis control programmes. Traditionally, detection of larvae is attained by the dissection of flies, but this time-consuming method cannot easily discriminate between species of Onchocerca. The genome of all Onchocerca species has a unique 150 bp repeat, which can be amplified by PCR, and O. volvulus-specific DNA probes can detect these products by Southern blot (SB). This study optimizes a PCR/SB assay, and compares it with fly dissection to estimate the prevalence (p) and intensity of infection (m) in the local vector population of a Mexican community that has become hypoendemic as a result of 7 years of treatment with ivermectin and nodulectomy. The PCR detected 1 infected fly in a pool of 99 uninfected flies, but the optimal pool size was 50 flies. At the community level, 1 out of 10550 flies was positive (p=0·0095%, 95% confidence intervals CI=0·00024–07·05280%; m=0·00027 larvae/parous fly, CI=−0·00026–0·00081) by PCR, and 4 out of 10772 flies (p=0·0371%, CI=0·01012–0.09505%; m=0·00107 larvae/parous fly, 95% CI=0·00002–0·00212) by dissection (observed m=0·0005). Both methods produce statistically similar estimates of the prevalence and intensity, indicating that pool screening is a viable alternative for entomological surveillance in areas where the intensity of transmission is becoming extremely low as a result of control interventions.

The survival capacities of Muellerius capillaris (Nematoda: Protostrongylidae) free-living larval stages (L1) of an Israeli desert isolate (Nubian isolate) (92 mm rain per annum) and a French isolate from a temperate habitat (650 mm rain per annum) were compared under humid and extreme dry conditions. Under the dry conditions (7% relative humidity [r.h.] and 33% r.h. at 23 °C), both isolates exhibited the same remarkable survival capacity for periods of 17 and 28 days, respectively (>92% survival, P>0·1). However, during and after recovery from the anhydrobiotic state, different behaviour patterns of L1 were observed in each isolate. Under humid conditions (97% r.h.) for 10 days, both isolates exhibited similarly low survival percentages (13·4±4·6%, S.E., survival for the Nubian isolate, 3±3% for the French isolate, P>0·05). In water, the French isolate was less active, survived better (52±8·4% vs 28·7±3·7, by day 28, P<0·05) and retained a better morphological appearance (shrunk less) than the Nubian isolate. Larval development of the French isolate in the land snail Theba pisana was significantly faster than that of the Nubian isolate, reaching a higher proportion of infective stages (L3) on day 36 post-infection (78·6% vs 56% P>0·05). However, similar infection intensities were recovered in snails infected with either of the isolates (59·8±10·3, S.E., n=14, for the French isolate, 53±6, n=13, for the Nubian isolate, P<0·05).

The excretory–secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 μg/ml of ESA protein (P<0·03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P<0·04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.

The aim of this work was to study the structure of the parasite communities of Digeneans of 2 families of Teleost fishes (Sparidae and Labridae) of the Mediterranean sea. We tried to quantify the importance of both the microhabitat requirements of the parasite species and the effect of host biological factors on the parasite communities. We applied, for the first time in parasite community studies, the Canonical Correspondence Analysis (CCA) to analyse (i) the spatial distribution of parasite species within the digestive tract of the hosts; (ii) the host's biological factors (such as diet, host length, gregariousness and abundance) that may influence this spatial distribution of parasite species. Our results showed that potential microhabitats were vacant in the 2 host families studied revealing a lack of niche saturation because either there was little inter- and/or intraspecific competition or there were enough available space and resources within the host. Our results also indicated that the position of the parasite in the digestive tract is much more important than host biological factors for the structure of parasite community. Finally, we highlight the potential use of the CCA method for controlling for phylogenetic constraints in multi-species analyses.

Cement gland protein in male and inseminated female individuals of an acanthocephalan parasite of fish, Pomphorhynchus laevis (Müller, 1776), was localized by immunohistochemistry using an antibody specific for cement protein. Male P. laevis possess 3 pairs of round to oval cement glands ranging from 0·5 to 0·9 mm in length and from 0·3 to 0·7 mm in width. Each gland has an outer portion containing nuclear fragments and other cellular organelles surrounding a space for storage of gland products. Very little work has been carried out on the nature of the cement gland secretions. We have previously reported that the major component of cement is a protein with molecular weight of 23 kDa; in fresh glands it is white in colour. Immunohistochemical studies herein reported were carried out using a polyclonal antibody raised against purified P. laevis p23 cement protein (anti-p23PL). Localization of p23 cement protein at the light microscope level, by means of the anti-p23PL antibody, shows that p23 is present within the cytoplasmic layer of the gland as well as in the gland duct lumen. Interestingly, the p23 cement protein was also identifiable at the posterior ends of females retaining the cap. Positivity to anti-p23PL antibody was obtained not only in the external part of the copulatory cap, but also within the vaginal tract and at the base of the uterine duct. Thus, we report herein the first photographic evidence that the copulatory cap is not a simple gonopore lid but it is really an intravaginal plug.

The somatic muscle of Ascaris suum is principally under the excitatory control of neuromuscular junction transmitter, acetylcholine (ACh). However, it has recently been shown that neuropeptides also play an important role in the motor-nervous system and one of these, AF3 (AVPGVLRFamide), also contracts muscle. The events which trigger contraction to ACh and AF3 would appear to be different, with ACh activating a nicotinic acetylcholine receptor whilst the response to AF3 is most likely to involve a G-protein coupled receptor negatively coupled to adenylate cyclase. In order to further elucidate differences in the cellular signalling pathways through which ACh and AF3 elicit muscle contraction, we investigated the actions of protein kinase C inhibitors, tamoxifen and chelerythrine, on the dorsal somatic muscle strip of A. suum. Contractions in response to 1 μM AF3 were potentiated by 17% in the presence of 10 μM tamoxifen (P<0·05; n=8); however, contractions in response to 10 μM ACh were markedly inhibited (tamoxifen IC50 44±18 μM; n=6). Tamoxifen also blocked muscle cell depolarizations to 5 μM ACh (IC50 4±1 μM; n=6) and 1 μM levamisole (IC50 14±6 μM; n=4). This was unlikely to be a non-specific effect on the membrane as hyperpolarizations to 10 μM GABA were unaffected (93% of control with 10 μM tamoxifen; n=6; P>0·05). However, another inhibitor of mammalian protein kinase C, chelerythrine, did not affect the response either to ACh or AF3 (n=6).

Authors' correction

In the recently published paper by J. Leiro et al. Parasitology119, 267–272, the legend to Fig. 4 should have read as follows:

Fig. 4. Dot–blot hybridization analyses to evaluate sensitivity of the probes C38-DIG (for Microgemma caulleryi) and F9-DIG (for Tetramicra brevifilum). Lanes 1 and 3, genomic DNA of M. caulleryi and T. brevifilum respectively; lanes 2 and 4, C38 and F9 fragments in pBluescript II SK(+). Visualization was with the aid of alkaline-phosphatase-coupled anti-DIG antibody (see text).