Biomedical subjects
Publications and source records attributed to P F Boreham.
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.
Five Giardia stocks collected from animals and man in Alberta, Canada, were compared by DNA fingerprinting. Although many DNA bands were common to all stocks, differences in the DNA banding patterns were seen. These same stocks had previously been shown to be identical by restriction enzyme cleavage of genomic DNA.
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.
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The development of an assay to measure the sensitivity of drugs against Blastocystis hominis using the incorporation of 3H-hypoxanthine is described. The activity of 42 compounds have been measured. Four of the 5-nitroimidazoles tested (satranidazole, S75 0400 A, flunidazole and ronidazole) were found to be more active than metronidazole, the drug commonly used to treat infections caused by B. hominis in humans. Other potentially useful compounds include emetine, furazolidone and quinacrine. Ketoconazole and iodoquinol reported to have therapeutic activity in infections caused by this parasite were found to be significantly less active than metronidazole.
The amitochondrial human intestinal parasite Giardia intestinalis is regarded to be the most ancient living example of single-celled eukaryotes and should display primitive features of pre-metazoan gene regulation. Characterization of E. coli clones which express Giardia antigens from plasmid vectors has revealed that an antigen is encoded by the rDNA repeat unit from the strand complementary to that encoding the rRNAs. The open reading frame (ORF) originates in the spacer region between the small (SS) and large (LS) subunit rRNA genes and terminates within the LS rRNA gene. The promoter region of this ORF has characteristics of both RNA polymerase (pol) II and pol III regulatory sequences, suggestive of gene regulation before these different promoter types evolved. The rDNA repeat unit is located on multiple chromosomal sites which are different in each isolate, although the electrophoretic karyotypes appear very stable in Giardia from both human and animal sources.
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A DNA fingerprint procedure has been established for the intestinal parasite, Giardia duodenalis. This permits the identification of individual strains from both human and animal sources. Analysis of strain movement, resurgence and variation is now possible. The fingerprint probe is based on the tandem repetitive sequence found in bacteriophage M13 which hybridizes to a set of hypervariable polymorphic minisatellite sequences found in higher eukaryotes. This probe recognizes a weakly homologous set of hypervariable regions in the Giardia genome to provide a DNA fingerprint comparable to those seen in higher eukaryotes.
Since the first description of Giardia by Antony van Leeuwenhoek in 1681, this parasite has proved to be enigmatic with a much more complicated epidemiology than originally envisaged. Even the nomenclature of this organism is confused. Despite numerous community prevalence studies, it was not until 1970 that the first proven report of waterborne transmission occurred. In 1981 the first common source outbreak due to contaminated food was recorded. It is also now recognized that venereal transmission occurs, especially among homosexual males. The faecal-oral route still remains the most important mode of infection despite the elucidation of these other methods of transmission. Recent developments in molecular biology especially DNA 'fingerprinting' and karyotyping which allow individual strain identification are being used to answer key epidemiological questions; for example are there truly pathogenic strains of Giardia, does drug resistance develop in vivo and is giardiasis a zoonotic disease? These and other molecular biological approaches will form the basis of research into the epidemiology of giardiasis and other intestinal protozoal infections until the end of the century and are certain to hold many surprises.
Evidence for drug resistance in giardiasis is reviewed and biochemical studies undertaken to determine the basis for this resistance are discussed. Metronidazole and furazolidone, which produce toxic radicals within the cell, have different biochemical mechanisms of action. Resistance to metronidazole is negatively correlated with the intracellular concentration of pyruvateferredoxin oxidoreductase leading to a concomitant decrease in the uptake of free metronidazole into the cell, while resistance to furazolidone appears to be due to an increase in thiol cycling enzymes. At the molecular level resistance to metronidazole is associated with DNA changes. DNA probes which hybridize with specific chromosomes and repetitive sequences indicate that rearrangements both at the chromosome and repetitive DNA level occurred concurrently with the development of metronidazole resistance. The problems of cross-resistance and treatment failures that occur in the absence of resistance are additional difficulties which have important implications for the management of individual patients. New drugs such as azithromycin, while showing great variation in activity against different stocks may be useful in treating some refractory cases of giardiasis. In the community, it is important to recognize the occurrence and spread of drug resistant Giardia, and markers, such as DNA probes, provide methods to monitor potential epidemics and the spread of drug resistant Giardia.
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In 1912 Blastocystis hominis was identified as a new species and classified as a yeast (Brumpt 1912). In the early 1920s several groups confirmed its classification as a yeast, specifically a member of the genus Schizosaccharomyces (discussed by Zierdt et al. 1967). Apart from an occasional case report, the classification of B. hominis and its role as a harmless intestinal yeast was not questioned for another 50 years. Then, Zierdt (1967) suggested that it should be classified in the phylum Protozoa, subphylum Sporozoa, and that it should be considered as a potential pathogen. The likely role of B. hominis as a human pathogen has recently become more firmly established (Garcia et al. 1984; Sheehan et al. 1986) and its classification has been changed. Although the classification of B. hominis as a protozoon was assumed widely, classification as a sporozoon was not accepted, and the most recent definitive classification of the Protozoa did not even list B. hominis (Lee et al. 1985). Then, based essentially on a review of the known characteristics of the organism, it was recently reclassified into the subphylum Sarcodina (Zierdt 1988). Clearly, the phylogeny of this emerging human pathogen needs definitive analysis (Mehlhorn 1988).
The technique of allozyme electrophoresis was applied to 29 Australasian stocks and 48 clones of Giardia intestinalis from humans as a means of increasing the number of genetic markers currently available for identification and classification. Fifty different enzymes were examined and of these 26 loci were found to be suitable for use as genetic markers. The data indicate the presence of four discrete genetic groups within the sample of G. intestinalis examined. The groups had fixed genetic differences at 23-69% of loci established. The evidence suggests that G. intestinalis is a species complex. The results have important implications for the systematics of human isolates of Giardia, as well as for studies on the epidemiology and demography of giardiasis in Australia and elsewhere.
An ultrastructural study of 10 different Blastocystis hominis stocks was undertaken. Three distinct morphological forms, vacuolar, granular and amoeboid, were distinguished. Numerous variations in the organelles and general cell structure were observed between stocks. B. hominis displayed considerable size variation in the vacuolar forms, ranging from 4 to 63 micron. Thickness and density of the surface coat varied between different stocks. Beneath the surface coat the bilaminar cell membrane displayed electron-dense pits. The nature and quantity of the vacuolar contents varied, and in the granular form four morphologically different inclusions were seen. The organelles which showed the greatest variation between stocks were the mitochondria, varying in shape, electron-density, type of cristae and presence of inclusions. There was minimal variation between stocks with regard to endoplasmic reticulum, Golgi complex and nuclei. Budding of material between the cytoplasm and central vacuole was observed in some stocks. Indications of phagocytic behaviour of B. hominis were seen in the amoeboid form and in the vacuolar form of one stock.
A total of 37 Giardia stocks isolated from humans and 14 stocks derived from animal sources have been analysed for antigenic differences. Separation of the proteins of the stocks by polyacrylamide gel electrophoresis showed no major differences among the stocks. Immunoblotting of these antigens demonstrated some minor differences which were not correlated with geographic location, allozyme type, virulence or any other distinguishing characteristic of the stocks. Immunofluorescence tests using monoclonal antibodies revealed some differences between stocks but the monoclonal antibodies did not significantly inhibit growth in inhibition assays.
A study of the function of the electron-dense pits in the vacuolar and granular forms of Blastocystis hominis was undertaken. Immuno-electron microscopy using anti-clathrin antibody and colloidal gold demonstrated clathrin to be associated with all forms of the pits and some cytoplasmic vesicles. Cationized ferritin traced the pathway of endocytosis from the surface of the coated pits through internalization via electron-dense coated vesicles and uncoated vesicles and tubules in the cytoplasm. The cationized ferritin particles accumulated in the central vacuole, suggesting a metabolic or storage role for this structure. Differences in the accumulation of cationized ferritin particles were noted between vacuolar and granular forms. The hydrolytic enzyme acid phosphatase was not detected within the central vacuole suggesting that this structure does not act as a lysosome.
Chromosomes of 41 stocks of Giardia duodenalis derived from humans and 14 stocks from other animal species were analysed by field inversion gel electrophoresis (FIGE). These stocks have two predominant karyotypes as judged by FIGE which appear to fit a geographic distribution. Under FIGE conditions used to optimize the detection of size variation in Giardia chromosomes, five or six major chromosomes could be identified. Most of the stocks derived from North America have three major chromosomes smaller than 800 kb while most of the Australian stocks have four. A few exceptions, and minor variations, of these karyotypes were observed. It was estimated that not all of the DNA entered the gel, the remainder being trapped conformations or very large chromosomes. Karyotypes of Giardia stocks from different animal hosts and human sources within a geographical region are similar.
Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to greater than 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.