P.F.L. Boreham

Giardia intestinalis: Electrophoretic evidence fora species complex

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.

Blastocystis in Humans and Animals: Morphology, Biology, and Epizootiology

Publisher Summary This chapter reviews the biology and epizootiology of Blastocystis. The various experimental attempts to induce different forms of Blastocystis hominis (B. hominis) in culture are discussed. The morphology of B. hominis is presented as determined by various techniques, such as light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Binary fission is the only proven method of reproduction for B. hominis. B. hominis cells dividing by binary fission are seen commonly by light microscopy and less frequently by electron microscopy. The cells divide into two approximately equal portions, with the distribution of organelles into both halves. The description of various forms of Blastocystis is discussed. Some of these forms include forms seen in vivo, fresh faecal form, cyst form, vacuolar form, granular form, and amoeboid form. The various Blastocystis spp. that exist in invertebrates, amphibia, reptiles, birds, and a variety of mammals, including rats, guinea pigs, cats, monkeys, and apes, are also reviewed.

Resistance to the nitroheterocyclic drugs

The nitroheterocyclic drugs have been available since the early 1960s for the treatment of anaerobic protozoa. The application of these drugs has widened since then and they are presently used to treat anaerobic pathogenic bacteria and protozoa. The activity of the nitroheterocyclic drugs depends on the all-important nitro group attached to the imidazole or furan ring. Although the nitro radicals, generated by reduction of the parent drugs, are similar for both families of nitroheterocyclics, the nitroimidazoles and the nitrofurans, the electron potential of each is different and thus the mechanism of action depends on different pathways. The nitroimidazoles depend on reduction by ferredoxin or flavodoxin. The nitrofurans require nitroreductase activity, but the natural substrate of these enzymes has not been identified. Increased use of nitroheterocyclic drugs, in response to drug resistance to other commonly used antibiotics, has in turn resulted in drug resistance to a number of nitroheterocyclic drugs. Bacteroides strains and other bacteria, including Helicobacter, have developed resistance. Among the protozoa, Trichomonas has developed resistance to metronidazole via a number of mechanisms, especially a decrease in drug reduction, as a result of alterations in the electron transport pathways. Resistance to both types of nitroheterocyclic drugs has been reported in Giardia. Although resistance to these drugs is not widespread, their increased use world-wide as a prophylaxis and in chemotherapy will inevitably result in increased resistance in organisms commonly found in asymptomatic infections, including Trichomonas, Giardia and Entamoeba. However, the variety of substitutions which can be attached to the ring structures has led to a great variety of drugs being synthesised, some of which are many-fold more active than the commonly prescribed nitroheterocyclics. With careful administration of currently available drugs and continued interest in synthesising more active compounds, we can optimistically expect to have useful nitroheterocyclic drugs available for some time.

Ultrastructural variation of Blastocystis hominis stocks in culture

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.

Altered uptake of metronidazole in vitro by stocks of Giardia intestinalis with different drug sensitivities

Growth of Giardia intestinalis in TYI-S-33 medium containing a sub-lethal concentration of metronidazole over a period of 66 weeks selected a line of organisms that was over 8 times less sensitive to the drug. This resistance was unstable and the organisms reverted to their original sensitivity within 22 weeks of growth in the absence of drug. A comparison of the uptake of [14C]metronidazole by the original and selected lines showed a highly significant reduction in drug uptake by the resistant line, indicating either a defective transport mechanism across the cell membrane or decreased reduction of metronidazole to the active metabolite within the cell. There was no significant difference in the uptake of metronidazole by 9 recent isolates of G. intestinalis maintained in axenic culture.

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.

Drug resistance in Giardia intestinalis

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.

Geographic variation in Giardia karyotypes

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.

Similarities of Giardia antigens derived from human and animal sources

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.

Chromosomes of Blastocystis hominis

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.