A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

A survey of Blastocystis in domestic chickens.

The prevalence and morphology of Blastocystis in fresh faecal material from 227 domestic chickens was investigated. A very high prevalence of infection (approximately 95%) was found in chickens from four of the five commercial farms studied. Extremely high numbers of Blastocystis were found in the majority of samples. Blastocystis cells showed considerable variation in size, ranging from approx. 3 microm to approx. 120 microm in diameter. This size range is more extreme than those previously recognised for the organism from chickens. All chickens from one farm appeared free of Blastocystis infection. Most Blastocystis cells appeared to be the vacuolar form, although the shape of the cells and the appearance of the central vacuole contents varied considerably within and among faecal samples. Nuclei showed "spots" of electron-opaque material, generally arranged as a band within the nuclei. Multiple individual cysts within a single outer fibrillar layer were found in addition to single cysts without an encompassing fibrillar layer.

Reappraisal of vesicular types in the syncytial tegument of the Echinococcus granulosus protoscolex.

The structure, abundance, and distribution of tegumentary vesicles was compared among Echinococcus granulosus protoscoleces that had been prepared for electron microscopy using four processing schedules: a conventional method, alternative fixations using uranyl acetate or osmium tetroxide-potassium ferricyanide, and a freeze-substitution method. Four morphologically distinct types of vesicles were found in the somal region. The morphology of the first form, with moderately electron-opaque contents, and the second form, with similar size and shape but containing an electron-opaque core, varied little among the preparation methods. Two additional forms of vesicles, with characteristic intensely electron-opaque contents, were revealed only after freeze-substitution. These elongate vesicles were also found in the scolex tegument where they were most conspicuous, and appeared markedly increased in number after freeze-substitution. Large, spherical vesicles with an electron-lucent core embedded in a dense matrix of fibrillar strands were the dominant vesicle forms in the scolex region after all methods of preparation. Fixation by osmium tetroxide-potassium ferricyanide revealed the presence of spherical vesicles with amorphous electron-opaque contents and a few inclusions. This form of vesicle was also observed after freeze-substitution, but the inclusions in the vesicular lumen were more numerous. The variation in the distribution of vesicle forms among the body regions strongly implies a variety of vesicle functions. In addition, our observations suggest that comparative studies of different fixative methods are necessary to demonstrate the detailed vesicular morphology of the tegument of E. granulosus and other cestodes.

Incidental finding of Myxobolus spores (protozoa: myxozoa) in stool samples from patients with gastrointestinal symptoms.

Myxozoan spores were detected in fecal samples from three patients presenting with abdominal pain and/or diarrhea. The spores were identical to those of Myxobolus plectroplites, a previously described pathogen from the freshwater fish Plectroplites ambiguus. All patients had recently eaten fish caught from local waters, and frozen fillets of such fish were found to be infected with M. plectroplites cysts. The passage of spores unchanged through the alimentary tract suggests they were incidental findings unrelated to clinical symptoms, especially since other enteric pathogens were present in two patients.

Immunolocalisation of the glutathione S-transferases, GST-26 and GST-28, within adult Schistosoma japonicum.

This paper describes the first ultrastructural immunolocalisation study of the 26-kDa and 28-kDa glutathione S-transferases within adult Schistosoma japonicum (GST-26 and GST-28). Polyclonal antibodies raised against GST-28 (in mice) and against GST-26 (in rabbits) were used to examine the distribution of the proteins within adult parasites. Both proteins were localised within the parenchymal region of the male parasite. Additionally, both proteins were present within parenchymal cells located between the vitelline glands of female parasites. There were no detectable levels of GST-26 or GST-28 on the surface or within the tegument matrix of either the male or female worms. Possible functions for GST-26 and GST-28 within S. japonicum and their significance as vaccine target molecules are addressed.

Ultrastructural analysis of the adult Schistosoma japonicum by lectin cytochemistry.

Eight lectin probes were used to detect a range of carbohydrate residues in the tegument matrix of Schistosoma japonicum. In addition, other areas of the parasite, such as the gut, vitelline glands and flame cells, were examined for carbohydrate residues. Some minor differences in the carbohydrate residue composition between tegument orientations and between the sexes were identified. Differences between the distribution of carbohydrate residues of S. japonicum examined in this study and previous reports of Schistosoma mansoni were also noted. This study further illustrates the high level of complexity within the tegument of the adult S. japonicum and has demonstrated differences between this species and the widely studied S. mansoni.

Detection of Blastocystis sp. in domestic dogs and cats.

Blastocystis sp. was detected in faecal samples from domestic dogs and cats in Brisbane, Australia. The prevalence rates were high, with 70.8% of the dogs and 67.3% of the cats infected with this organism. Blastocystis sp. from faecal material from two dogs was successfully cultured on inspissated egg slant medium for several months, but could not be maintained for longer periods. Blastocystis sp. from feline faecal samples failed to grow in culture. The parasites found in dogs and cats were generally smaller than Blastocystis hominis from human faecal material, and were the vacuolar form rather than the multivacuolar form. Otherwise, the general morphology of these organisms appeared similar to B. hominis when examined by light and transmission electron microscopy.

Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum.

This paper describes the first localization study of the 14.7 kDa fatty acid-binding protein in Schistosoma japonicum (SjFABPc) using transmission electron microscopy. A polyclonal antibody raised against recombinant Sj-FABPc was used in combination with a colloidal gold marker to determine the distribution of the protein within adult parasites. Sj-FABPc was localized within lipid droplets below the subtegumental region of the male parasite. Additionally, Sj-FABPc was present in the vitelline droplets of the vitelline glands of female parasites. There were no detectable levels of Sj-FABPc on the surface or within the tegument of male or female parasites. Possible functions of Sj-FABPc within S. japonicum and the relevance of these immunolocalization findings in light of the recent reports that the homologue Sm-FABPc is an important anti-S. mansoni vaccine target molecule are also discussed.

Virus-like particles in Blastocystis sp. from simian faecal material.

Virus-like particles were observed in the cytoplasm of Blastocystis sp. found in fresh faecal material from 2 monkeys (Macaca fascicularis). Two morphological types of particles were found during examination by transmission electron microscopy: 1 was approximately 55-60 nm diameter, with an electron-opaque core of approximately 30 nm in diameter; and the other was approximately 200 nm in diameter with a core of approximately 100 nm in diameter. This is the first report of virus-like particles identified in situ in Blastocystis.

Macromolecular transport in malaria--does the duct exist?

The proposal that a parasitophorous 'duct' traverses the malaria-infected erythrocyte cytoplasm and is responsible for the unusual molecular uptake kinetics observed in malaria, has created considerable debate on the nature of macromolecular transport in this parasite. The existence of a 'duct' has important implications for the immunobiology of this parasite, particularly the possibility that antibodies may have access to 'internal' antigens in malaria. The most compelling evidence that there is a direct connection between the parasite and the surrounding media comes from the experiment of Pouvelle et al. (Nature 353, 73-75 (1991)) using small highly fluorescent latex spheres. However, we have found that fluorescent labeling of the parasite and tubular structures that extend from the parasite is due to the release of dye from the latex spheres during the incubation and is not due to the uptake of the spheres themselves. The inability of malaria-infected erythrocytes to take up latex beads down to 14 nm diameter establishes that an 'open' channel connecting the parasite with the surrounding media does not exist. This finding has important implications for establishing the unusual nature of macromolecular transport across the infected erythrocyte cytoplasm in malaria.

Morphological differences in Blastocystis cysts-an indication of different species?

Cyst forms of Blastocystis that show disparate morphology in relation to the previously described cysts were detected in faecal material from animal hosts. Transmission electron microscopy was performed without attempts to isolate or concentrate Blastocystis from the faecal material. Large, multinucleate cyst forms were found in faecal material from Macaca monkeys. These cyst forms measured up to approximately 15 microm in diameter and were often larger than vacuolar forms present in the same samples. Four or more nuclei were frequently seen in the cysts. Multiple individual cysts enclosed by a single fibrillar layer were found in faecal material from domestic chickens. Each individual cyst within the multiple cyst form measured approximately 3-4 microm in diameter and appeared to be uninucleate.

Schistosoma japonicum: immunolocalization of paramyosin during development.

This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.

Blastocystis hominis revisited.

Blastocystis hominis is a unicellular organism found commonly in the intestinal tract of humans and many other animals. Very little is known of the basic biology of the organism, and controversy surrounds its taxonomy and pathogenicity. There morphological forms (vacuolar, granular, and ameboid) have been recognized, but recent studies have revealed several additional forms (cyst, avacuolar, and multivacuolar). The biochemistry of the organism has not been studied to any extent, and organelles and structures of unknown function and composition are present in the cells. Several life cycles have been proposed but not experimentally validated. The form used for transmission has not been defined. Infections with the organism are worldwide and appear in both immunocompetent and immunodeficient individuals. Symptoms generally attributed to B. hominis infection are nonspecific, and the need for treatment is debated. If treatment appears warranted, metronidazole is suggested as the drug of choice, although failures of this drug in eradicating the organism have been reported. Infection is diagnosed by light microscopic examination of stained smears or wet mounts of fecal material. Most laboratories identify B. hominis by observing the vacuolar form, although morphological studies indicate that other forms, such as the cyst form and multivacuolar form, also should be sought for diagnosis.

The ultrastructural localization of Echinococcus granulosus antigen 5.

Murine monoclonal and polyclonal antisera, raised against the 38 kDa subunit of Echinococcus granulosus antigen 5, were used to investigate the tissue distribution of the antigen in hydatid cysts. Immunoreactivity was visualized by indirect immunofluorescence on whole protoscoleces, and ultrastructural immunocytochemistry utilizing colloidal gold-based labelling procedures on unsectioned and cryosectioned brood capsules and protoscoleces. In protoscoleces, the 38 kDa subunit of antigen 5 was localized at the interface of parenchymal cells and associated extracellular matrices, as well as along the interface of the tegumentary syncytium in the somal region and its basal matrix. Cytoplasmic labelling of parenchymal cells was rare; when observed, it was associated with vesicles and membranes in cytoplasmic extensions of parenchymal cells. In brood capsules, the antigen was associated with the external face of the plasma of degenerating parenchymal cells. The 38 kDa subunit occurs along the extracellular face of cell membranes, suggesting that antigen 5 is either a component of the membranes or associated extracellular matrices.

Peptide polymerisation facilitates incorporation into ISCOMs and increases antigen-specific IgG2a production.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.

Bacteria-like endosymbionts in Blastocystis sp.

Bacteria-like endosymbionts were found in vacuolar cells and cysts of Blastocystis sp. from duck and monkey faecal material. The organisms ultrastructurally resembled Gram-negative bacilli, and were present in the nucleus of Blastocystis sp. from the duck and in the cytoplasm of Blastocystis sp. from the monkey. Based on size and differences in intracellular location, it is probable that these represent two distinct species of organisms.

Electron microscopy of surface structures of Blastocystis sp. from different hosts.

Samples of Blastocystis sp. obtained from humans, monkeys, pigs and chickens were examined by scanning electron microscopy and transmission electron microscopy to compare surface structures. The surface coat of Blastocystis sp. cells from each host species showed some morphological variations, but these were not sufficiently different to allow judgement to be made on speciation. The surface structure morphology appeared similar for samples of Blastocystis sp. from the same host species. The surface coat of the cultured human isolate of B. hominis was much thinner than that of cells from fresh human faecal material, and the cell surface appeared to be smoother and without the small projections seen in the fresh forms. Bacteria were frequently found in association with the surface coat of Blastocystis sp. from all fresh faecal material. Possible functions of the surface coat, especially in relation to protection against osmotic shock, are discussed.

Morphology of Blastocystis sp. from domestic birds.

A study of Blastocystis sp. from domestic birds was undertaken to determine if morphological differences occurred. Fresh faecal material from domestic chickens, ducks and geese and from commercially farmed ostriches was obtained. Blastocystis sp. from chickens was morphologically very different from that from the other hosts, having within the nucleus discrete spots of chromatin rather than a crescentic band (ducks and geese) or an elliptical band (ostrich). A thick surface coat surrounded all Blastocystis sp. cells in the faecal material, with isolates from the ostrich having the thickest surface coat relative to the cell diameter. Cysts were more commonly found in the chicken samples but were also detected in the duck and ostrich samples. This study suggests that three morphologically distinct groups are represented: one in the chicken, one in the ostrich and another in ducks and geese. These tentative conclusions require confirmation by molecular techniques.

Morphology of Blastocystis sp. isolated from circus animals.

Blastocystis sp. is reported for the first time from faecal samples collected from a camel, a llama, a highland bull and a lion in a travelling circus. Fresh faecal specimens were examined by light and electron microscopy, and vacuolar and cyst forms of similar morphology were present in all three ungulates. These cells were smaller than cultured vacuolar cells of Blastocystis hominis isolated from humans and contained only a single vacuole in comparison to the multivacuolar cell found in fresh human faeces. The taxonomic relationship of Blastocystis isolated from humans and ungulates remains to be determined. The number of parasites present in the lion sample was too small to make valid comparisons.