PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum Isolates of Human and Animal Origin

Abstract

Genomic DNAs from humanCryptosporidium spp. are Apicomplexan parasites that infect the gastrointestinal or respiratory tract of humans and animals. In immunocompetent hosts, the infection is typically acute and self limiting, whereas in immunocompromised individuals, such as persons receiving immunosuppressive drugs and AIDS patients, cryptosporidiosis is often a chronic disease. Since drug therapy to control or eliminate these organisms is not yet available, persistent infections in these patients are therefore especially severe and can be life threatening. The potential of Cryptosporidium as an opportunistic parasite and the recent reports of major outbreaks of cryptosporidiosis in the United States, United Kingdom, and Australia due to contamination of drinkable water supplies indicate that Cryptosporidium should be considered a major public health problem (12,25).To date, eight Cryptosporidium species have been regarded as valid on the basis of host specificity, pathogenesis, and oocyst morphology (13). These included Cryptosporidium parvum in mammals, Cryptosporidium muris in rodents and ruminants, Cryptosporidium felis in domestic cats, Cryptosporidium wrairi in guinea pigs, Cryptosporidium baileyi and Cryptosporidium meleagridis in birds, Cryptosporidium serpentis in reptiles, and Cryptosporidium nasorum in fishes. According to this classification, the causative agent of cryptosporidiosis in humans and a range of mammalian species is the species C. parvum. Numerous PCR-based assays have been described for detection of Cryptosporidium parasites. The primers of these PCR assays are based on either undefined genomic sequences (2, 3, 22, 27, 46) or specific genes (4, 27-29, 35, 40, 41, 43, 45, 48, 49). Most PCR assays have led to the confirmation of C. parvum as the major cause of cryptosporidiosis in humans and to the identification of two genotypes within this species: the "human" genotype (genotype 1), which has so far been found exclusively in humans, with the exception of a single nonhuman primate (39) and a dugong (33), and the "bovine" genotype (genotype 2), found in domestic livestock such as cattle, sheep, and goats, etc., which can also infect humans. Additional genotypes have then been distinguished in C. parvum (32). However, most of the available genotyping tools were designed to analyze clinical specimens, and their specificities for other C. parvum genotypes or other Cryptosporidium species were not always established.Laxer et al. were among the first authors to publish primers for detection of Cryptosporidium (22). These primers, which were not known to target a specific gene but to amplify a 452-bp fragment of an unidentified region, as well as the reported 452-bp sequence, have been widely used (1, 6-11, 14, 15, 17, 18, 20, 21, 23, 37, 38). The purpose of the present study was to investigate the extent of sequence heterogeneity for this diagnostic DNA fragment among human isolates of Cryptosporidium.Sample analysis. Fecal samples used in this study were obtained from infected bovine (named...

Prevalence ofCryptosporidiumspp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia

“…The sequence of the chickenderived isolate was identified as C. meleagridis. The Cryptosporidium DNA diagnostic fragment characterized by Laxer et al (Guyot et al, 2002;Laxer et al, 1991) was amplified by PCR for the broiler chickenderived isolate only. In agreement with the genotyping of the 18S rRNA gene, C. meleagridis was identified by RFLP (data not shown).…”

Cryptosporidium parvum(Eucoccidiorida: Cryptosporiidae) in calves: results of a longitudinal study in a dairy farm in Sfax, Tunisia

“…Amplified products were sequenced by using the BigDye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). In addition to this genotyping technique, DNA amplification and restriction fragment length polymorphism (RFLP) at the Laxer locus were performed as previously reported (Guyot et al, 2002). The nucleotide sequences generated in this study has been deposited in GenBank under accession number EF158462.…”

Molecular Characterization ofCryptosporidiumIsolates from Humans and Animals in Iran

“…All specimens were genotyped on the basis of the 18S rRNA gene by nested PCR-restriction fragment length polymorphism (RFLP) (25,26,28) and sequencing (8). Cryptosporidium species were further confirmed by a Laxer sequencebased tool as previously described (9). Indeed, distinct Laxer PCR-RFLP patterns allowed the differentiation of C. hominis, C. parvum, and C. meleagridis, even distinguishing between two subgenotypes of C. parvum, as a result of DNA variation within this species (9).…”