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.

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.

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.

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.

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.

Synthesis and assembly of infectious bovine papillomavirus particles in vitro.

Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

Bile acids influence the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells.

The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells.

Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles.

A recombinant vaccinia virus termed pLC201VV was designed to coexpress the L1 and L2 late genes of human papillomavirus type 16 (HPV16). Synthesis of the L1 and L2 proteins occurred in cells infected with pLC201VV, and 40-nm virus-like particles with a density of 1.31 g/ml were produced in the nuclei of cells synthesizing both L1 and L2, but not in cells synthesizing either protein alone. Virus-like particles were partially purified from infected cells by sucrose gradient sedimentation and shown to consist of capsomeres similar to HPV and contain glycosylated L1 viral capsid protein. The production of HPV-like particles using recombinant vaccinia virus should be useful for biochemical studies and could provide a safe source of material for the development of a vaccine.

Ultrastructure of Blastocystis hominis in human stool samples.

A study of the ultrastructure of Blastocystis hominis in human stools found morphological differences between the organisms seen and those present in laboratory cultures. B. hominis found in stool samples showed little morphological variation with storage time before fixation, but were consistently smaller (approximately 5 microns in diameter), with a thicker surface coat than the cultured organisms. The large central vacuole, characteristic of the cultured organisms, and accepted as standard morphology of B. hominis, was rarely observed in organisms present in stool samples. Instead, a number of small vacuoles, or possibly a network of interconnected vacuoles, were noted. After short-term culture, organisms from these samples appeared with the typical vacuolated morphology. No large vacuoles were present in organisms obtained at colonoscopy. These results suggest that the vacuolated form as previously described may be an artefact of culture conditions, and that the form of B. hominis present in the gastrointestinal tract is avacuolar.

A cyst-like stage of Blastocystis hominis.

A cyst-like form of Blastocystis hominis is described in stools and in culture. This form is more common in stored stools than fresh material. A cyst wall is secreted under the surface coat of the cell, and the surface coat and cell debris subsequently separate from the cyst. Whether this stage can withstand adverse environmental conditions and is infective to a new host remain to be determined.

Cellular localisation of tumour antigen (TA-4) in normal, dysplastic and neoplastic squamous epithelia of the upper aerodigestive tract.

We report the use of tumour antigen (TA-4) polyclonal antiserum to assess the level and pattern of TA-4 antigen expression in formalin-fixed paraffin-embedded tissue sections from 110 patients with a range of normal, dysplastic and malignant squamous epithelia from various sites in the upper aerodigestive tract. There was a high degree of TA-4 antigen expression in the superficial layers of normal squamous epithelium and in well-differentiated squamous cell cancers (SCC). TA-4 expression was consistently absent in dysplastic oral squamous epithelium and in poorly differentiated SCCs. The degree of cellular heterogeneity in moderately differentiated SCCs was such that morphologically identical squamous cancer cells could be distinguished on the basis of TA-4 expression. Immuno-electron microscopy localised TA-4 antigen to tonofibrils in both normal buccal (squamous) cells and in squamous cancer cells. Results of Western blotting confirmed the presence of a 48 kDa protein consistent with TA-4 antigen in both SCCs and in normal buccal mucosa. We conclude that TA-4 protein is a normal cellular component, that cellular TA-4 expression is related to the level of cellular differentiation in squamous epithelia and that it is not likely to be useful as an independent index of cellular proliferation or malignant behaviour.