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

摘要

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.