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) (ϳ300 bp) and second internal transcribed spacer (pITS-2) (ϳ230 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 ϳ1.3 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 ϳ1 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.Species of Cryptosporidium are oocyst-forming apicomplexan parasites which infect a wide range of vertebrate hosts. Intestinal disease caused by these parasites is called cryptosporidiosis and is transmitted via the fecal-oral route (e.g., in child day care centers) and can be associated with waterborne outbreaks (27). The clinical signs of cryptosporidiosis in humans are mainly diarrhea, dehydration, malabsorption, weight loss, and/or wasting (27). In many cases, infection is self-limiting and resolves spontaneously, although chronic infections may develop in high-risk patient groups, particularly neonates and immunocompromised individuals (those with congenital or acquired immunodeficiencies or those undergoing immunosuppressive chemotherapy). The accurate identification of Cryptosporidium to the species or "strain" (i.e., genotypic) level is important for studying transmission patterns and genetic structures and is central to the control of cryptosporidiosis (9, 26, 34). Currently, 11 species of Cryptosporidium are recognized (9, 10), of which C. parvum is of major significance in humans. However, these species cannot be distinguished from one another simply on the basis of host occurrence and/or parasite morphology (9, 27). Consequently, a...

Classification ofCryptosporidiumSpecies from Patients with Sporadic Cryptosporidiosis by Use of Sequence-Based Multilocus Analysis following Mutation Scanning

“…The relative specificity of these primers for the amplification of Cryptosporidium species by PCR has been demonstrated previously (24,49). The cycling conditions were one cycle of 94°C for 5 min (denaturation), followed by 35 cycles of 94°C for 30 s (denaturation), 58°C for 20 s (annealing), and 72°C for 30 s (extension), and then a final extension of 72°C for 7 min.…”

“…To identify the species of Cryptosporidium present in each DNA sample, an ϳ300-bp region of the small subunit of the nuclear rRNA (p-SSU) gene was amplified by PCR from genomic DNA (ϳ10 to 20 ng) using the oligonucleotide primers 18SiF (forward; AGTGACAAGAAATAACAATACAGG-3Ј) and 18SiR (reverse; 5Ј-CCTGCTTTAAGCACTCTAATTTTC-3Ј) (24,49) and subjected to sequencing (described below). The relative specificity of these primers for the amplification of Cryptosporidium species by PCR has been demonstrated previously (24,49).…”

“…However, rare genetic variants, such as C. hominis IaA17R1, C. hominis IfA12G1R2, and C. parvum IIaA22G4R1 (present study), indicate that localized, potentially geographically unique, subgenotypes are present even in large, urban populations in cosmopolitan cities, emphasizing the need for the increased study of underrepresented countries (such as those in Africa, Asia, and South America, as well as most island nations). There have been some suggestions (24,57) that strain variation within Cryptosporidium species is correlated with geographical origin. Also, various authors (1,5,7,51,84,87) have reported gp60 genotypes or subgenotypes that infect both humans and animals and have formulated hypotheses regarding the importance of zoonotic transmission in causing sporadic cases or outbreaks of cryptosporidiosis in humans.…”

“…The population genetic analysis of C. parvum and C. hominis has revealed intraspecific variability in host specificity and geographical distribution (6,24,33,51,82,84). For C. parvum, some of these intraspecific variants have been associated with substantial variation in minimum infective dosage and/or virulence (52,53).…”

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“…Even though Sanger sequencing is still considered as a "golden standard" for detection of unknown mutations, there is a need to develop and use new methods which can reduce cost and time needed for individual examinations. There are many methods that are capable of detecting changes in nucleotide sequence of DNA such as: SSCP (single-strand conformation polymorphism analysis) [28], DGGE (denaturing gradient gel electrophoresis) [29,30], TGCE (temperature gradient capillary electrophoresis) [31]. Until now high-resolution analysis of melting curves seems unparalleled (High Resolution Melting, HRM) [32].…”