Giovanni Widmer

The genome of Cryptosporidium hominis

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa—protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome ∼9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

A hundred-year retrospective on cryptosporidiosis

Tyzzer discovered the genus Cryptosporidium a century ago, and for almost 70 years cryptosporidiosis was regarded as an infrequent and insignificant infection that occurred in the intestines of vertebrates and caused little or no disease. Its association with gastrointestinal illness in humans and animals was recognized only in the early 1980s. Over the next 25 years, information was generated on the diseases epidemiology, biology, cultivation, taxonomy and development of molecular tools. Milestones include: (i) recognition in 1980 of cryptosporidiosis as an acute enteric disease; (ii) its emergence as a chronic opportunistic infection that complicates AIDS; (iii) acknowledgement of impact on the water industry once it was shown to be waterborne; and (iv) study of Cryptosporidium genomics.

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.

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.

Detection and Genotyping of Oocysts of Cryptosporidium parvum by Real-Time PCR and Melting Curve Analysis

ABSTRACT Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum β-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same β-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.

Infectivity of a Cryptosporidium parvum isolate of cervine origin for healthy adults and interferon-γ knockout mice

The infectivity of a Cryptosporidium parvum isolate of cervine origin (type 2, Moredun) propagated in calves was investigated simultaneously in healthy adult human volunteers and in interferon-gamma knockout (GKO) mice. After exposure to 100-3000 oocysts, 16 volunteers recorded, for a duration of 6 weeks, the number and form of stools that they passed and any symptoms that they experienced. Oocyst excretion was assessed by enzyme-linked immunosorbent assay and direct immunofluorescence assay. Eleven subjects (69%) became ill, and 8 subjects (50%) shed oocysts in stool. The median duration of illness was 169 h, and the median number of unformed stools passed was 24. The duration and intensity of symptoms were more severe than were those associated with previously studied isolates. The median infectious dose was estimated to be 300 oocysts for humans and 1 oocyst for the GKO mouse model. The Moredun isolate was more pathogenic than the reference GCH-1 isolate. The GKO mouse model of cryptosporidiosis is useful for discerning isolate-specific differences in pathogenicity.

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.

Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves.

Genotypic analyses of Cryptosporidium parvum oocysts have divided the species into two genotypes, referred to as type 1 and type 2. Although humans are susceptible to both types, mixed type 1/type 2 infections have rarely been identified. The paucity of mixed infections could be explained by the predominance of one type over the other in mixed infections, or by the poor sensitivity of restriction fragment length polymorphism (RFLP) analyses for detecting subpopulations. Using a type-specific real-time PCR assay capable of detecting type 1 or type 2 constituting as little as 0.01% of the population, archived and new isolates of human, bovine, and mouse origin were genotyped. Mixed type 1/type 2 infections were identified in humans and calves, including in samples previously found to be homogeneous by RFLP. Isopycnic fractionation of mixed isolates revealed that type 1 and type 2 oocysts differ in their sedimentation properties. The detection of a type 1 subpopulation in serially-propagated bovine isolates indicates that type 1 and type 2 are stably maintained during long-term passage. Together with recently reported experimental bovine and ovine type 1 infections, the persistence of type 1 subpopulation in experimentally infected animals suggests that animals may play a previously unrecognized role in the maintenance of C. parvum type 1.

Genetic Analysis of a Cryptosporidium parvum Human Genotype 1 Isolate Passaged through Different Host Species

ABSTRACT Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and β-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

A pig model of the human gastrointestinal tract

Easy access to next generation sequencing has enabled the rapid analysis of complex microbial populations. To take full advantage of these technologies, animal models enabling the manipulation of human microbiomes and the study of the impact of such perturbations on the host are needed. To this aim we are developing experimentally tractable and clinically relevant pig models of the human adult and infant gastro-intestinal tract. The intestine of germ-free piglets was populated with human adult or infant fecal microbial populations, and the piglets were maintained on solid or milk diet, respectively. Amplicons of 16S rRNA V6 region were deep-sequenced to monitor to what extent the transplanted human microbiomes changed in the pig. Within 24 h of transfer of human fecal microbiome to pigs, bacterial microbiomes rich in Proteobacteria emerged. These populations evolved toward a more diverse composition rich in Bacteroidetes and Firmicutes. In the experiment where infant microbiome was used, the phylogenetic composition of the transplanted bacterial population converged toward that of the human inoculum. A majority of sequences belonged to a relatively small number of operational taxonomic units, whereas at the other end of the abundance spectrum, a large number of rare and transient OTUs were detected. Analysis of fecal and colonic microbiomes originating from the same animal indicate that feces closely replicate the colonic microbiome. We conclude that the pig intestine can be colonized with human fecal microbiomes to generate a realistic model of the human GI tract.