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G Widmer

Publications and source records attributed to G Widmer.

At least 19 recordsLinked to original sources
PubMed2001

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Animals↗
PubMed2000

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Animals↗
PubMed2000

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

Animals↗
PubMed2000

Host cell apoptosis impairs Cryptosporidium parvum development in vitro.

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.

Animals↗
PubMed1999

beta-tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability.

Determining the viability of waterborne Cryptosporidium parvum oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The beta-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in C. parvum known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4 degrees C for over 39 weeks maintained their infectivity and displayed no decrease in the level of beta-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of C. parvum oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.

Animals↗
PubMed1999

Constitutive expression of small subunit ribosomal RNA transcripts in Cryptosporidium parvum oocysts and intracellular stages.

As in other apicomplexan protozoa, the ribosomal genes of Cryptosporidium parvum are present in small number and dispersed on several chromosomes. Two types of ribosomal small subunit genes have been identified in bovine isolates by sequencing and sequence-specific polymerase chain reaction. Using denaturing gradient gel electrophoresis, ribosomal gene expression was investigated in C. parvum oocysts and intracellular stages grown in tissue culture. In a calf-propageted C. parvum isolate, extra and intracellular stages expressed 2 types of transcripts. This observation contrasts with the differential expression of rRNA, genes in malaria species. Analysis of ribosomal gene expression in oocysts of a different genotype indicates that some C. parvum isolates express only l type of small subunit ribosomal RNA. These observations highlight fundamental differences in ribosomal gene expression among apicomplexan protozoa and within the species C. parvum and support the view that differential expression in malaria is an adaptation to a dixeneous life cycle.

Animals↗
PubMed1998

Genetic heterogeneity and PCR detection of Cryptosporidium parvum.

A variety of methods have been applied to the study of genotypic and phenotypic polymorphism in Cryptosporidium parvum. Results from these studies have consistently shown the existence of different genotypes and phenotypes within the species. A long-term goal of this work is the identification of markers for virulence in humans and animals and the elucidation of transmission cycles of C. parvum. Achievement of these goals will depend on the identification of highly polymorphic loci. Of particular interest are polymorphisms amenable to typing by polymerase chain reaction (PCR), as C. parvum cannot be expanded in vitro. Fingerprinting of isolates by restriction of PCR fragments or allele-specific PCR has given promising results. As originally observed by isoenzyme analysis, genetic fingerprinting has confirmed the occurrence in humans of unique C. parvum genotypes which are not found among calf isolates. This observation remains to be reconciled with the cross-infectivity of C. parvum to ruminant and nonruminant hosts and the important role that bovines play in the epidemiology of C. parvum and human cryptosporidiosis. Although PCR detection of C. parvum DNA from individual oocysts has been reported, the sensitivity of PCR detection when working with environmental or fecal samples is significantly reduced. Therefore, PCR is currently not used for routine diagnosis or environmental monitoring for C. parvum. Inhibitors present in environmental samples, mainly in water and soil, which can negatively affect PCR recoveries, have been identified, and several methods have been proposed to circumvent these problems. The further refinement of detection and genetic fingerprinting protocols will provide essential tools for indentifying environmental sources of oocysts and elucidating transmission cycles.

Animals↗
PubMed1998

Genotypic and phenotypic characterization of Cryptosporidium parvum isolates from people with AIDS.

Genotypic analysis of Cryptosporidium parvum has demonstrated the presence of two subgroups within the species, whereas biochemical and antigenic characterization have shown more heterogeneity. The clinical relevance of these observations is unknown. C. parvum isolates from people with AIDS were studied with respect to parasite genotypes and virulence in cell monolayers and laboratory animals. Ten of 13 oocyst samples had a characteristic human-associated (H) genotype; 3 had a genotype typical of calf-excreted oocysts (C). Virulence in cell culture was mildly or markedly lower in the 5 isolates tested (4 H and 1 C) compared with the GCH1 reference isolate. H isolates did not infect newborn ICR mice, whereas 1 of the 2 C isolates tested did. These findings reinforce the concept of C. parvum genetic subgroupings that correlate to some extent with infectivity and suggest that additional heterogeneity is present within the subgroups.

AIDS-Related Opportunistic Infections↗
PubMed1998

Sequence polymorphism in the beta-tubulin gene reveals heterogeneous and variable population structures in Cryptosporidium parvum.

Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the beta-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the beta-tubulin intron and adjacent exon 2. Isolates carrying different beta-tubulin alleles were found. Significantly, one of the beta-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different beta-tubulin alleles fluctuated during serial passage.

Animals↗
PubMed1998

Transmission and serial propagation of Enterocytozoon bieneusi from humans and Rhesus macaques in gnotobiotic piglets.

For over a decade Enterocytozoon bieneusi infections in people with AIDS have been linked with chronic diarrhea and wasting. The slow scientific progress in treating these infections is attributed to the inability of investigators to cultivate the parasite, which has also precluded evaluation of effective therapies. We report here successful serial transmissions of E. bieneusi from patients with AIDS and from macaques with AIDS to immunosuppressed gnotobiotic piglets. One infected piglet was still excreting spores at necropsy 50 days after an oral challenge. Spores in feces were detected microscopically by trichrome stain and by PCR and within enterocytes by in situ hybridization and immunohistochemistry. E. bieneusi infection induced no symptoms. The development of an animal model for E. bieneusi will open up new opportunities for investigating this parasite.

AIDS-Related Opportunistic Infections↗
PubMed1998

Paromomycin and geneticin inhibit intracellular Cryptosporidium parvum without trafficking through the host cell cytoplasm: implications for drug delivery.

Cryptosporidium parvum, which causes intractable diarrhea and lethal wasting in people with AIDS, occupies an unusual intracellular but extracytoplasmic niche. No reliable therapy for cryptosporidiosis exists, though the aminoglycoside paromomycin is somewhat effective. We report that paromomycin and the related compound geneticin manifest their major in vitro anti-C. parvum activity against intracellular parasites via a mechanism that does not require drug trafficking through the host cell cytoplasm. We used both normal and transformed aminoglycoside-resistant Caco-2 or MDBK cells in these studies. Timed-exposure experiments demonstrated that these drugs inhibit intracellular but not extracellular parasites. Apical but not basolateral exposure of infected cells to these drugs led to very significant parasite inhibition, indicating an apical topological restriction of action. We estimated intracytoplasmic concentrations of paromomycin, using an intracellular bacterial killing assay, and found that C. parvum infection did not lead to increased paromomycin concentrations compared to those in uninfected cells. Global [3H]paromomycin uptake by Caco-2 cells was approximately 200-fold higher than the estimated intracytoplasmic paromomycin concentration, suggestive of host cell vesicular uptake and concentration (as has been reported with other cell lines). However, preinfection exposure of Caco-2 cells to paromomycin did not result in subsequent inhibition of parasite development, indicating that if exogenous paromomycin enters the infected host cell vesicular compartment, it does not effectively communicate with the parasite. Thus, the apical membranes overlying the parasite and parasitophorous vacuole may be the unsuspected major route of entry for paromomycin and may be of importance in the design and discovery of novel drug therapies for the otherwise untreatable C. parvum.

Animals↗
PubMed1998

Multilocus genotypic analysis of Cryptosporidium parvum isolates from different hosts and geographical origins.

The genetic analysis of oocysts recovered from the stools of humans and animals infected with Cryptosporidium parvum has consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis, C. parvum isolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species C. parvum.

Animals↗
PubMed1997

Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts.

Cryptosporidium parvum is an enteric protozoan parasite of medical and veterinary importance. Dissemination of environmentally resistant oocysts in surface water plays an important role in the epidemiology of cryptospridiosis. Although the polymerase chain reaction (PCR) is a well-established technique and is widely used for detecting microorganisms, it is not routinely applied for monitoring waterborne C. parvum. In order to facilitate the application of PCR to the detection of waterborne C. parvum oocysts, a comparison of published PCR protocols was undertaken and different sample-preparation methods tested. The sensitivity of a one-step PCR method, consisting of 40 temperature cycles, was 10 purified oocysts or fewer than 100 oocysts spiked in raw lake water. The detection limit of two primer pairs, one targeting the ribosomal small subunit and another specific for a C. parvum sequence of unknown function, was approximately ten-fold lower than achieved with a primer pair targeting an oocyst shell protein gene. Three cycles of freezing/thawing were sufficient to expose oocyst DNA and resulted in higher sensitivity than proteinase K digestion, sonication or electroporation. Inhibition of PCR by surface water from different local sources was entirely associated with the soluble fraction of lake water. Membrane filtration was evaluated in bench-scale experiments as a means of removing lake water inhibitors and improving the detection limit of PCR. Using gel and membrane filtration, the molecular size of inhibitory solutes from lake water was estimated to less than 27 kDa.

Animals↗
PubMed1997

Transmission and establishment of a persistent infection of Enterocytozoon bieneusi, derived from a human with AIDS, in simian immunodeficiency virus-infected rhesus monkeys.

Enterocytozoon bieneusi is closely linked with chronic diarrhea and wasting in AIDS. Although reported >10 years ago, little is known about the infection and the disease it induces in humans. Duodenal E. bieneusi spores from an AIDS patient were orally transmitted to 2 simian immunodeficiency virus-infected rhesus monkeys. Both animals began shedding spores within a week of inoculation, as confirmed by microscopy and polymerase chain reaction, and continued until euthanatized 7 and 8 months later. E. bieneusi infection in the gut was sparse, either because of moderate numbers of circulating CD4 cells or because monkeys are less susceptible than humans to this infection. This is apparently the first documented transmission of E. bieneusi infection between hosts.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients.

The microsporidium Enterocytozoon bieneusi is closely linked to wasting and diarrhea in a high proportion of individuals with AIDS. However, its relative contribution to disease is uncertain because diagnosis until recently depended on procedures involving endoscopy. A sensitive PCR technique which amplifies a fragment of the small-subunit rRNA gene of E. bieneusi from formalin-fixed stool samples was developed. Of 80 formalin-fixed stool samples collected from 74 Zimbabweans and 6 U.S. patients who were human immunodeficiency virus positive, 50% tested positive for E. bieneusi by PCR, whereas 24% tested positive for E. bieneusi by light microscopy of trichrome-stained fecal smears. In addition, we describe an in situ hybridization technique which detected and identified E. bieneusi as the causative agent in all six intestinal biopsy specimens tested. Both the PCR and in situ hybridization procedures are sensitive diagnostic tools which will complement currently available techniques and enable the differentiation of E. bieneusi from other microsporidia to be made.

AIDS-Related Opportunistic Infections↗
PubMed1997

A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts.

Length and restriction site polymorphism within a 2.8-kb threonine-rich open reading frame from Cryptosporidium parvum was identified and used to determine the genotypes of isolates from calves and humans. In agreement with observations of other genetic loci, all calf isolates were identical at this locus. In contrast, human isolates showed two profiles, one found exclusively in humans and one a superposition of both profiles, which were indicative of heterogeneous parasite populations. PCR fingerprints were consistent with a change in the genetic profile of C. parvum isolates following transmission from bovine to human hosts.

Amino Acid Sequence↗
PubMed1996

Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat.

Oocysts of the protozoan parasite Cryptosporidium parvum are found in most surface waters and can contaminate municipal water supplies, as demonstrated by recent outbreaks of cryptosporidiosis. A method capable of fingerprinting C. parvum isolates from the environment would facilitate the study of epidemiology and transmission cycles and aid in the implementation of preventive measures to reduce water contamination by oocytes. In this study, we report polymorphism in C. parvum isolates on the basis of analysis of random amplified polymorphic DNA and nucleotide sequences in a region of the 18S rRNA and the internal transcribed spacer 1. Isolate-specific primers for these two regions were designed, and PCR tests capable of discriminating between isolates were developed. In both PCR assays, the five C. parvum isolates analyzed segregated into two subgroups. One group consisted of isolates that originated directly from human patients, and the other group had various host origins and had been propagated in laboratory animals. These results demonstrate the feasibility of distinguishing C. parvum isolates by sequence-specific PCR tests.

Animals↗