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The CpA135 gene as a marker to identify Cryptosporidium species infecting humans
Fabio Tosini1, Rosanna Drumo, Kristin Elwin
1Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. fabio.tosini@iss.it
Abstract:
Of the 22 species currently recognized as valid in the Cryptosporidium genus, C. parvum and C. hominis account for most cases of human infections worldwide. However, C. meleagridis, C. canis, C. felis, C. suis, C. muris, as well as the cervine, rabbit and monkey Cryptosporidium genotypes, have also been recognized as the etiologic cause of both sporadic and epidemic cryptosporidiosis in humans. Molecular methods are necessary to distinguish species and genotypes of Cryptosporidium, due to the lack of reliable morphological variations. The aim of this work was to determine the genetic polymorphisms in a fragment of the A135 gene in isolates of C. parvum, C. hominis, C. meleagridis, C. canis, C. muris, C. andersoni and the Cryptosporidium cervine genotype. Primers were designed on conserved regions identified on a multiple alignment of the C. parvum, C. hominis and C. muris sequences, the three species for which information is available at the genome level. PCR amplification and direct sequencing of a 576 bp fragment revealed the presence of numerous single nucleotide polymorphisms (SNPs) among the species/genotype tested. The genetic variability was exploited to design a PCR-RFLP assay useful for a rapid identification of the most important human pathogens in the genus Cryptosporidium.
Insights
This study identifies genetic variations in the Cryptosporidium A135 gene across various species and genotypes. These findings enable a new PCR-RFLP assay for quickly identifying major human Cryptosporidium pathogens.
Area of Science:
- Medical Parasitology
- Molecular Biology
- Genetics
Background:
- Cryptosporidium is a significant cause of human gastrointestinal illness, with Cryptosporidium parvum and Cryptosporidium hominis being the most prevalent species.
- Several other Cryptosporidium species and genotypes, including C. meleagridis, C. canis, C. felis, C. suis, C. muris, and cervine, rabbit, and monkey genotypes, are also implicated in human cryptosporidiosis.
- Distinguishing between Cryptosporidium species and genotypes is crucial for understanding transmission and epidemiology, but reliable morphological differences are lacking, necessitating molecular methods.
Purpose of the Study:
- To investigate genetic polymorphisms within a specific fragment of the A135 gene in various Cryptosporidium species and genotypes.
- To develop a molecular tool for the rapid and accurate identification of clinically relevant Cryptosporidium species and genotypes.
Main Methods:
- Design of primers targeting conserved regions of the A135 gene based on available genomic data for C. parvum, C. hominis, and C. muris.
- PCR amplification of a 576 bp fragment of the A135 gene from isolates of C. parvum, C. hominis, C. meleagridis, C. canis, C. muris, C. andersoni, and the cervine genotype.
- Direct sequencing of the amplified DNA fragments to identify single nucleotide polymorphisms (SNPs).
- Development of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay based on identified genetic variations.
Main Results:
- The study successfully amplified and sequenced a 576 bp fragment of the A135 gene from the tested Cryptosporidium isolates.
- Numerous single nucleotide polymorphisms (SNPs) were identified among the different species and genotypes, highlighting significant genetic variability.
- The identified genetic polymorphisms were effectively utilized to create a novel PCR-RFLP assay.
Conclusions:
- The A135 gene fragment exhibits sufficient genetic variability to differentiate between various Cryptosporidium species and genotypes.
- The developed PCR-RFLP assay provides a rapid and efficient method for identifying major human Cryptosporidium pathogens.
- This molecular tool can aid in epidemiological studies and clinical diagnostics of cryptosporidiosis.