Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species

Abstract

Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and beta-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

FIG. 1.

COWP PCR-RFLP (A) and 5B12 microsatellite (B) analyses of oocysts from the four C. parvum genotype 1 isolates passaged through different host species. (A) UG502 human (lane 2), pig (lane 3), and calf (lane 4) passages; UHP5 human (lane 5), pig (lane 6), and calf (lane 7) passages; TPH1 human (lane 8) and pig (lane 9) passages; and TU502 human (lane 10), pig (lane 11), and calf (lane 12) passages. A genotype 2 control (lane 14), genotype 1 control (lane 15), uncut PCR product (lane 16), and 100-bp DNA ladder (lanes 1, 13, and 17; Promega Corp., Madison, Wis.) are also included. (B) UG502 human passage (lane 1), UG502 calf passage (lane 2), UHP5 human passage (lane 3), UHP5 pig passage (lane 4), TPH1 human passage (lane 5), TU502 human passage (lane 6), and TU502 calf passage (lane 7). GCH1 (genotype 2) and NEMC1 (genotype 1) controls are shown in lanes 8 and 9, respectively.

FIG. 2.

Genotype-specific PCR analysis of genotype 1 isolates passaged through different host species. (A) Each sample was assayed using the genotype 1-specific primers to demonstrate the absence of PCR inhibitors. (B) The presence of a genotype 2 subpopulation was assayed using the TRAP C1 genotype 2-specific primers. Samples are as follows: human isolate UG502 (lane 2), UG502 pig passage 1 (lane 3), UG502 pig passage 9 (lane 4), UG502 isolate from calf 3 (lane 5), UHP5 human (lane 6), UHP5 pig passage 1 (lane 7), UHP5 isolate from calf 1 (lane 8), UHP5 isolate from calf 2 (lane 9), human isolate TPH1 (lane 10), TPH1 pig passage 1 (lane 11), human isolate TU502 (lane 12), TU502 pig passage 1 (lane 13), TU502 pig passage 6 (lane 14), TU502 pig passage 11 (lane 15), TU502 pig passage 18 (lane 16), TU502 isolate from calf 1 (lane 17), TU502 isolate from calf 2 (lane 18), TU502 isolate from calf 3 (lane 19), TU502 isolate from calf 4 (lane 20), TU502 isolate from calf 9 (lane 21), and TU502 isolates calves 11 and 12 (lane 22). Genotype standards include genotype 1 (lane 23), mixed genotype 1 and 2 (lane 24), and genotype 2 (lane 25). DNA ladder (100 bp; Promega Corp.) is shown in lanes 1 and 26. A faint band is seen in TU502 isolate from calf 2 (lane 18) using the genotype 2-specific primers, indicating the presence of genotype 2 parasites.