Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom

ABSTRACT Cryptosporidium parvum oocyst DNA samples (n = 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (∼300 bp) and second internal transcribed spacer (pITS-2) (∼230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of C. parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of ∼1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C. parvum type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (n = 20; one child nursery outbreak) and type 2 (n = 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of ∼1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.

[1]  A. Crisanti,et al.  PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. , 2006, FEMS microbiology letters.

[2]  J. Rose,et al.  Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites. , 2002, Journal of microbiological methods.

[3]  J. Isaac-Renton,et al.  Novel Cryptosporidium Genotypes in Sporadic Cryptosporidiosis Cases: First Report of Human Infections with a Cervine Genotype , 2002, Emerging infectious diseases.

[4]  R. Chalmers,et al.  Infection with unusual types of Cryptosporidium is not restricted to immunocompromised patients. , 2002, The Journal of infectious diseases.

[5]  R. Fayer,et al.  CRYPTOSPORIDIUM CANIS N. SP. FROM DOMESTIC DOGS , 2001, The Journal of parasitology.

[6]  R. Gasser,et al.  Applications of single-strand conformation polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes. , 2001, Veterinary parasitology.

[7]  E. Dei‐Cas,et al.  Molecular Characterization ofCryptosporidium Isolates Obtained from Humans in France , 2001, Journal of Clinical Microbiology.

[8]  E. Pozio,et al.  Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum. , 2001, International journal for parasitology.

[9]  M. M. Marshall,et al.  Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts , 2001, Applied and Environmental Microbiology.

[10]  R. Gasser,et al.  Mutation scanning analysis of sequence heterogeneity in the second internal transcribed spacer (rDNA) within some members of the Hypodontus macropi (Nematoda: Strongyloidea) complex , 2001, Electrophoresis.

[11]  R. Gasser,et al.  Genotyping Cryptosporidium parvum by single‐strand conformation polymorphism analysis of ribosomal and heat shock gene regions , 2001, Electrophoresis.

[12]  R. Gasser,et al.  High‐resolution electrophoretic procedures for the identification of five Eimeria species from chickens, and detection of population variation , 2000, Electrophoresis.

[13]  S J Upton,et al.  Epidemiology of Cryptosporidium: transmission, detection and identification. , 2000, International journal for parasitology.

[14]  S. Tzipori,et al.  Extensive Polymorphism in Cryptosporidium parvumIdentified by Multilocus Microsatellite Analysis , 2000, Applied and Environmental Microbiology.

[15]  E. Pozio,et al.  A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum , 2000, Parasitology.

[16]  R. Fayer,et al.  Variation in Cryptosporidium: towards a taxonomic revision of the genus. , 1999, International journal for parasitology.

[17]  M. Abrahamsen,et al.  Microsatellite analysis of the human and bovine genotypes of Cryptosporidium parvum. , 1999, The Journal of eukaryotic microbiology.

[18]  R. Gasser,et al.  Isolation, propagation and characterisation of Cryptosporidium. , 1999, International journal for parasitology.

[19]  P. Deplazes,et al.  Sequence and PCR–RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts , 1999, Parasitology.

[20]  P. Guldberg,et al.  Analysis of sequence homogenisation in rDNA arrays of Haemonchus contortus by denaturing gradient gel electrophoresis , 1998, Electrophoresis.

[21]  R. Gasser,et al.  Single‐strand conformation polymorphism (SSCP)‐based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes , 1998, Electrophoresis.

[22]  R. Gasser,et al.  A mutation scanning approach for the identification of hookworm species and analysis of population variation. , 1998, Molecular and biochemical parasitology.

[23]  S. L. Le Blancq,et al.  Ribosomal RNA gene organization in Cryptosporidium parvum. , 1997, Molecular and biochemical parasitology.

[24]  R. Gasser,et al.  Mutation scanning methods for the analysis of parasite genes. , 1997, International journal for parasitology.

[25]  U. Morgan,et al.  Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. , 1997, The Journal of parasitology.

[26]  R. Gasser,et al.  Identification of parasitic nematodes by PCR-SSCP of ITS-2 rDNA. , 1997, Molecular and cellular probes.

[27]  S. Tzipori,et al.  Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat , 1996, Applied and environmental microbiology.

[28]  J. Elder,et al.  Concerted Evolution of Repetitive DNA Sequences in Eukaryotes , 1995, The Quarterly Review of Biology.

[29]  G A McConkey,et al.  The cytoplasmic ribosomal RNAs of Plasmodium spp. , 1995, Parasitology today.

[30]  P J O'Donoghue,et al.  Cryptosporidium and cryptosporidiosis in man and animals. , 1995, International journal for parasitology.

[31]  Christian Schlötterer,et al.  Chromosomal homogeneity of Drosophila ribosomal DNA arrays suggests intrachromosomal exchanges drive concerted evolution , 1994, Current Biology.

[32]  T. McCutchan,et al.  Structurally distinct, stage-specific ribosomes occur in Plasmodium. , 1987, Science.

[33]  J. Dame,et al.  Two major sequence classes of ribosomal RNA genes in Plasmodium berghei. , 1984, Nucleic acids research.

[34]  Peter H. A. Sneath,et al.  Numerical Taxonomy: The Principles and Practice of Numerical Classification , 1973 .

[35]  P. Hunter,et al.  Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. , 2002, International journal for parasitology.

[36]  G. Widmer.  Genetic heterogeneity and PCR detection of Cryptosporidium parvum. , 1998, Advances in parasitology.