Host mucins have crucial physical roles in preventing the parasitic establishment and maturation, and also in expelling the invading parasites. However, some parasites utilize mucinase enzymes to facilitate the infection. Recently, we have identified a mucinase enzyme of the liver fluke Opisthorchis viverrini, Ov-M60-like-1, which exhibits metallopeptidase activity against bovine submaxillary mucin substrate. Here, we aimed to study the localization of this enzyme in O. viverrini and the bile duct of hamsters using immunohistochemistry and functional analysis by mucin digestion in hamsters and mice tissues. The results showed that Ov-M60-like-1 was detected strongly in the tegument, tegumental cells, vitelline glands and mature eggs with miracidium. Expression in the gut, ovary and testis of the parasite was moderate while parenchyma showed slight colour intensity. In addition, the mucinase was also detected in the host biliary epithelial cells and goblet cells surrounding the worm. The mucinase assay revealed that the Ov-M60-like-1 could digest neutral mucin in the parenchyma, testis and seminal receptacle, but not the mucin in the tegument, tegumental cells and vitelline glands of the worm. The enzyme can also digest mucin in the cholangiocytes and modified the mixture type in the bile duct goblet cells of the infected hamsters, a susceptible host. In contrast, the enzyme was unable to digest neutral, acid and mixture mucin in the bile duct of the mice, a non-susceptible host. These findings indicate that Ov-M60-like-1 may have functions in both housekeeping tasks and host–parasite interactions, especially in modification of host susceptibility.

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named ‘megasomes’. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

The A subunit of NF-Y nuclear factor from Schistosoma mansoni was expressed in E. coli fused to a histidine tag and purified by affinity chromatography using a Ni2+-Agarose matrix. Antibodies against the recombinant protein were prepared and used for Western blot and immunolocalization. The presence of SMNF-YA in all stages of the parasite life-cycle was determined by RT-PCR and Western blot analysis. The immunolocalization of SMNF-YA showed the presence of this factor in a parenchymal cell population of cercariae and adult worms and in embryos within eggs. The expression of SMNF-YA was demonstrated to decrease in maturating spermatozoites whereas an accumulation of this factor was observed in the nucleus from oocytes during their maturation processes.

The cuticle of filarial nematodes is a specialized extracellular matrix that covers the parasite and protects it from adverse conditions of the environment. As a surface structure it is in direct contact with the host defence mechanisms and therefore plays an important role in the molecular host-parasite relationship. Using polyclonal antisera raised against the insoluble components of the cuticle of the adult filarial parasite Brugia pahangi, we have isolated cDNA clones encoding collagen molecules of the cuticle. The protein domain structure of cDNA clone Bpcol-1 was compared with the known structures of cuticular collagens of the nematodes Brugia malayi, Caenorhabditis elegans, Ascaris suum and Haemonchus contortus, confirming interspecies similarities. Using affinity-purified anti-Bpcol-1 antibodies we identified Bpcol-1 antigenic determinants in different nematode extracts, and determined the localization of such epitopes within the cuticle of B. pahangi.

Secretions of plant-parasitic nematodes which are released into plant tissue may play critical roles in plant-nematode interactions. The identification and characterization of these molecules are of fundamental importance and may help to facilitate the development of novel strategies to interfere with nematode infection of plants and thereby decrease nematode-induced damage to crops. An antibody-based approach was used to isolate molecules present on the nematode surface and in nematode secretions. Monoclonal antibodies (MAbs) were produced to secretions and to whole Heterodera avenue 2nd-stage juveniles; several of these MAbs recognized molecules present in nematode secretions produced in vitro. Three of these molecules have been partly characterized in H. avenae, Globodera rostochiensis, G. pallida and Meloidogyne incognita. A MAb reacting with the surfaces of these nematodes recognized antigens of different molecular weight in each of the species tested. This difference in antigenicity might be related to specific functions in these nematodes. Preliminary results show that this antibody also localized the antigen in root cells surrounding the feeding site induced by M. incognita in Arabidopsis thaliana.

We cloned and expressed Echinostoma caproni HSP70 in Escherichia coli. This molecule presents an open reading frame (ORF) of 655 amino acids, and a theoretical molecular weight of 71 kDa. E. caproni HSP70 protein showed a high homology to other helminth molecules, major differences being located in the C-terminal region of the molecule, with a hydrophobic portion. Studies of protein and messenger RNA (mRNA) expression revealed a distinct pattern, depending on the host (low- or high-compatible). Specific polyclonal antisera raised against the recombinant protein expressed in Escherichia coli demonstrated its selective presence in excretory/secretory products (ESP) of adult parasites obtained from high-compatible hosts. Immunological studies showed clearly the association of HSP70 with the parasite surface and other structures, including eggs.

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster.

The acrosome reaction is a fundamental event in the biology of the sperm and is a prerequisite to fertilization of the egg. Members of the Rho family of GTPases and their effectors are present in the cytoplasm and/or plasma membrane overlying the acrosome of porcine sperm. We have implicated the Rho family of GTPases and the Rho-activated kinase, ROCK-1, in mediating the zona-pellucida-induced acrosome reaction. Others have implicated the Rho GTPase in regulating the ionophore-induced acrosome reaction in the sperm of several mammalian species as well as in motility of bovine sperm. In this study, the localization of the Rho GTPases (RhoA, RhoB, Rac1 and Cdc42) as well as the effectors RhoGDI, PI(4)P5K and ROCK-1, was determined in boar, human, rat, ram, bull and elephant sperm. The four GTPases were each present in the sperm head of all species examined. RhoGDI was expressed in the head and tail of sperm from all species except pig, where it was present only in the head. PI(4)P5K was expressed in both head and tail of sperm from all species, but expression was typically weaker in the tail. Finally, ROCK-1 was expressed in the heads and tails of all sperm except that of the boar, where it was present only in the acrosomal region. These observations taken together suggest that the expression of Rho GTPases in sperm has been conserved throughout mammalian evolution, most likely due to the role of these GTPases in regulating acrosomal exocytosis.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

During asexual development Plasmodium schizonts undergo a series of complex biochemical and structural changes. Using tightly synchronized cultures of 2 P. falciparum lines (clone C10 and strain ITO4) for light microscopy and fluorescence imaging we monitored the timing and sequence of expression of proteins associated with invasion-related organelles. Antibodies to rhoptry, micronemal and dense granule proteins (Rhoptry Associated Protein 1, Apical Membrane Antigen 1, Erythrocyte Binding Antigen 175, Ring-infected Erythrocyte Surface Antigen) and to pellicle-associated proteins (Merozoite Surface Protein 1, PfMyosin-A) were used. Clone C10 developed faster than ITO4; this difference was also found in the timing of protein expression seen by immunofluorescence. Light microscopic data were combined with transmission electron microscopic analysis using serial sectioning of ITO4 schizonts to determine nuclear number and organellar development. Thus a timetable of schizont structural maturation was established. Generally, the timing of organelle-specific antigen expression correlates well with the ultrastructural data. Rhoptries are formed mainly between second and fourth nuclear divisions, micronemes between the end of the fourth nuclear division and merozoite separation from the residual body, while dense granules are generated mainly after the micronemes. PfAMA-1 appears in micronemes before EBA-175, suggesting micronemal heterogeneity.

The fact that the Schistosoma mansoni egg has two ATP diphosphohydrolase (EC 3.6.1.5) isoforms with different net charges and an identical molecular weight of 63000, identified by non-denaturing polyacrylamide gel electrophoresis and immunological cross-reactivity with potato apyrase antibodies, is shown. In soluble egg antigen (SEA), only the isoform with the lower net negative charge was detected and seemed to be the predominant species in this preparation. By confocal fluorescence microscopy, using anti-potato apyrase antibodies, the S. mansoni egg ATP diphosphohydrolase was detected on the external surface of miracidium and in von Lichtenberg's envelope. Intense fluorescence was also seen in the outer side of the egg-shell, entrapped by the surface microspines, suggesting that a soluble isoform is secreted. ATP diphosphohydrolase antigenicity was tested using the vegetable protein as antigen. The purified potato apyrase was recognized in Western blots by antibodies present in sera from experimentally S. mansoni-infected mice. In addition, high levels of IgG anti-ATP diphosphohydrolase antibodies were detected by ELISA in the same sera. This work represents the first demonstration of antigenic properties of S. mansoni ATP diphosphohydrolase and immunological cross-reactivity between potato apyrase and sera from infected individuals.

The 14-3-3 protein is a key player in signal transduction processes in various species. We have previously cloned and expressed the 14-3-3 of Schistosoma mansoni. Using the purified protein we have now raised antibodies against it. A highly specific, affinity-purified antibody preparation was employed for the localization of the 14-3-3 protein in the parasite, by both immunohistochemistry and immunoelectron microscopy. The results demonstrate wide distribution of this protein. It was observed in the female excretory system, the nephridia as well as in the genital systems of both sexes, namely in the vitelline gland of female and in the testis of the male. It is also present in the parenchyma and muscle of both male and female worms. Immunoelectron microscopy demonstrated the presence of immunogold-labelled protein in the tegument, subtegument, muscle, parenchyma and in the female reproductive system, in both the cytoplasm and nucleus of vitelline cells, and oocytes. The possible role of the 14-3-3 protein in the genital organs is discussed.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatinNucleotide and amino acid sequence data reported in this paper are available in the GenBankTM database under the accession number AF035945.. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.

A schistosome infection is initiated when the parasite penetrates the skin of a susceptible host. Relatively large quantities of protein are released by transforming cercariae compared to later larval stages. This represents the first parasite material to which the host's immune system is exposed, yet little is known about the proteins which are released during the first few hours post-transformation. We have shown that antiserum raised against such molecules was capable of imparting protection against a schistosome challenge infection upon passive transfer to naïve mice. By screening a cercarial cDNA library with this serum, 38 positive clones were identified. Sequence analysis showed these to represent 8 different molecules which included Schistosoma mansoni 21·7 kDa antigen, calcium-binding-protein and the vaccine candidate glutathione S-transferase (Sm28GST). In addition, 5 clones were isolated, 1 of which had significant homology to many cytochrome C proteins, another with leukocyte elastase inhibitors and 3 which represented novel molecules. Four clones were expressed in a prokaryotic high-level expression vector, sera produced against each purified recombinant protein and used subsequently to probe Western blots and parasite sections. The leukocyte elastase inhibitor homologue and 2 unknowns induced significant proliferation by lymph node cells recovered from mice vaccinated with irradiated cercariae. More strikingly, the 2 novel proteins stimulated very high levels of interferon γ (IFNγ) secretion both by lymph node cells and those recovered by broncho-alveolar lavage from the lungs of vaccinated mice. Such results will be discussed in the context of vaccine development.

Schistosoma mansoni depends for its survival on glycolysis. Two glycolytic enzymes, glyceraldehyde-3P-dehydrogenase and triose-phosphate dehydrogenase, found in both the adult and schistosomular tegument, have been reported to confer partial protection against cercarial infection. This paper describes the immunogenic properties of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis, and its localization in the tegument and adjacent tissues. Recombinant schistosome PFK was used as antigen. A polyclonal antibody against purified PFK from Fasciola hepatica was affinity purified using recombinant PFK and used in combination with immunogold labelling to identify PFK by transmission electron microscopy in cryosections. In both adult worms and in schistosomula most immunogold label localized in the cytoplasmic syncytial region with less being found in the tegument. There was no significant PFK localization within or external to the outer membrane. Sera from mice immunized with recombinant S. mansoni PFK with Freund's adjuvant or alum plus rIL-12 demonstrated high titres of anti-PFK IgG, but no protection against cercarial infection. Sera from mice that were acutely or chronically infected or multiply exposed to irradiated cercariae did not recognize recombinant schistosome PFK in either Western blotting or ELISA. Similarly, sera from humans infected with S. mansoni did not recognize PFK. We conclude that in spite of the high immunogenicity of rPFK in mice, it is not a significant immunogen during the course of infection and does not confer protection from schistosomiasis. One main difference between PFK and the other 2 glycolytic enzymes seems to be the inaccessibility of PFK to the outside surface of the tegument.

A gene encoding a protein of 646 amino acid residues with a molecular mass of 71·3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb). The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum. Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage). Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm. The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella.

Lung-stage schistosomula are the target of protective immunity in mice vaccinated with attenuated cercariae of Schistosoma mansoni. Therefore, proteins present at this developmental stage, and in particular those which are secreted, are a potential source of novel vaccine candidates. However, little information is available about such molecules. Here we describe the cDNA clones identified by screening expression libraries with serum raised against proteins released by lung-stage schistosomula. In total, 11 different cDNA species were identified, 6 of which have been described previously in S. mansoni; these included fructose 1,6-bisphosphate aldolase and Sm21.7 which together accounted for two-thirds of all positive clones. Of the 5 newly described schistosome genes, 1 cDNA had a high degree of homology to the s5a subunit of 26S proteasomes, most significant being with the human protein. The remaining 4 clones showed no significant homologies to any genes sequenced previously. Fructose 1,6-bisphosphate aldolase, Sm21.7, the proteasome homologue and 1 unknown clone (A26) have been expressed in a bacterial expression system and serum produced against each recombinant protein. Immunolocalization showed fructose 1,6-bisphosphate aldolase, Sm21.7 and the proteasome homologue to be most abundant in muscle cells whilst clone A26 was distributed throughout many tissues, but was most abundant in the tegument. Analysis of the cellular immune responses of vaccinated mice showed 3 of the 4 expressed clones to be highly immunogenic, inducing the secretion of large quantities of the Th1-type cytokine interferon gamma.

Murine antibodies, raised against a purified recombinant 30 kDa laminin-binding protein from Echinococcus granulosus, were used to investigate the tissue distribution of the native protein in protoscoleces and brood capsules. Immunofluorescence, in combination with confocal microscopy, revealed that the protein was distributed in small annular foci near the peripheral regions of the protoscoleces. Immunoelectron microscopy of thawed cryosections demonstrated that the laminin-binding protein was present in the cytoplasm of tegumentary cytons and myocytons, but not in cells of the excretory system. The protein was associated with amorphous regions of the cytoplasm, and was not expressed at the surfaces of cells. This distribution resembles those of other invertebrate laminin-binding proteins, which are thought to act in the cell cycle and cell proliferation events. A low degree of label was consistently detected in extracellular matrices of the protoscolex.