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.

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.

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.

Variation in protein concentrations in acute and chronic ascites in Dirofilaria immitis-infected dogs.

Microfilaremic dogs, developing ascites acutely following a reaction to diethylcarbamazine therapy, had similar protein concentrations in their ascitic fluid and plasma. In contrast, in dogs chronically infected with Dirofilaria immitis, the protein concentrations of ascitic fluid were found to be significantly lower than plasma protein concentrations. The acute development of ascites in such dogs is associated with high ascitic protein levels.

Dreamtime, devastation and deviation: Australia's contribution to the chemotherapy of human parasitic infections.

There are three areas in which Australian scientists have made outstanding contributions to the study of the chemotherapy of human parasitic infections. Naturally occurring products of plants have great potential as antiparasitic agents and although several native species have been shown to have antimalarial and anthelmintic activity, their potential as chemotherapeutic agents has not been fully realised; secondly, the demands of war ensured that the Army Malaria Unit at Cairns carried out meticulous and exceptional studies to evaluate new antimalarial compounds. Not only were they able to prove the effectiveness of atebrin, Proguanil and chloroquine as prophylactics, they also obtained much new information on the pharmacokinetics of antimalarials and about the infection itself. Full recognition of these pioneering studies involving over 1000 volunteers infected with malaria, which can never be repeated, has not been appreciated. The third significant contribution is the molecular studies on the mechanisms of drug resistance Plasmodium falciparum of both the antifolate- and quinoline-containing drugs and the identification and subsequent biochemical and molecular analysis of drug resistance in Giardia intestinalis infections.

Biological and genetic analysis of a longitudinal collection of Giardia samples derived from humans.

Duodenal aspirates from children investigated for diarrhoea have been examined for the presence of Giardia over an eleven year period, and where possible, in vitro or in vivo Giardia cultures in mice were established. Based on biochemical characteristics of electrophoretic karyotype, RFLP analysis and rDNA hybridization studies of 40 stocks at least two major varieties, or demes, of Giardia have infected the population of South East Queensland and environs during this period. These demes carried different rDNA repeat units and differed markedly in both the electrophoretic karyotype pattern and the molar representation of chromosome bands. From 1983 to 1991 only one deme was documented. The first evidence of a new deme seen in local children occurred in 1991 and was followed by a predominance of this deme in 1993. These 40 stocks do not represent all positive samples. Other stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks were derived from children with similar chronic symptoms it appears that at least two demes of Giardia are pathogenic.

Resistance to the nitroheterocyclic drugs.

The nitroheterocyclic drugs have been available since the early 1960's 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.

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.

Oxygen affinities of metronidazole-resistant and -sensitive stocks of Giardia intestinalis.

The common protozoon, Giardia intestinalis, parasitizes the upper small intestine of man, and is often refractory to treatment by metronidazole. Defective oxygen-scavenging mechanisms have been implicated as a cause of metronidazole resistance of another flagellate Trichomonas vaginalis, where metronidazole is also the most common drug treatment. Oxygen consumption of six clinical isolates of G. intestinalis and one line selected for resistance to metronidazole was measured over 0-50 microM-O2 using an oxygen electrode open for gas exchange. At > 30 microM-O2, inhibition of respiration was demonstrated in all seven stocks. Apparent oxygen affinities (KmO2) were found to range from 0.5 to 5.2 microM-O2; however, isolates from patients who failed to respond to treatment with metronidazole did not have measurably defective O2-scavenging capabilities compared with metronidazole-sensitive isolates. These strains did, however, show elevated NADPH-oxidase activities compared with metronidazole-sensitive strains. Results indicate that biochemical mechanisms of drug resistance in G. intestinalis may be quite different from those operating in T. vaginalis.

A gene associated with cell division and drug resistance in Giardia duodenalis.

A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650-800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range. The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock. G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA. Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4. The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite. In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture. Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

Changes in allozyme pattern of the protozoan parasite Giardia intestinalis.

The present study compares the allelic profiles of Giardia intestinalis grown in vivo and in vitro. Three clinical isolates of G. intestinalis were established in suckling mice and subsequently adapted to in vitro culture to test the null hypothesis that samples of the same clinical isolate grown in different culture conditions have identical allelic profiles. For each isolate, a mouse-derived and an axenically cultured sample were analysed electrophoretically at 11 enzyme loci. In each case, the axenically cultured sample of each isolate showed marked allelic differences from its corresponding in vivo sample. These data suggest that there may be either regulated expression of alternative genes encoding distinct isozymes (i.e. gene switching) or selection by different growth conditions of specific genotypes from a mixture present within the original clinical isolate. Although these hypotheses are not tested in this study, the data highlight the importance of confirming that allozymes (or isozymes) are stable genetic characters for the identification and characterization of protozoan taxa.

Protein and DNA evidence for two demes of Blastocystis hominis from humans.

Analysis of 10 stocks of Blastocystis hominis isolated from human stools revealed two discrete groups of organisms. Proteins of the two groups were immunologically distinct and hybridization with random probes generated from the DNA of one stock showed that the DNA content of the two groups was different. Further studies are required to determine whether these should be classified as discrete species or whether these groups are epidemiologically significant.