Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis

Abstract

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

FIG. 1

Electrophoretic analysis of 5B12 and 1G09 microsatellites. DNA was amplified from the isolates shown in the presence of 5′ ³²P-labeled reverse primer, resolved on a sequencing gel, and visualized by autoradiography. (A) 5B12 locus; (B) 1G09 locus. Genotype 2 isolates are italicized. The host origins of GCH1, MD, and UCP oocysts are indicated in panel B. The positions of selected markers from a 10-bp ladder are shown at the right. neg., negative PCR control.

FIG. 2

Sequence analysis of cloned PCR products originating from 1F07 and 5B12 loci. One clone was picked randomly for each isolate and sequenced in both orientations. The locations of forward (F) and reverse (R) primers are shown with arrows. Genotype 2 isolates are italicized. Periods (.), deletions; tildas (∼), not sequenced.

FIG. 3

Genotypic profile of four genotype 2 isolates passaged from calves to GKO mice. Oocysts originating from calves (lanes 1, 3, 5, and 7) and GKO mice (lanes 2, 4, 6, and 8) were genotyped in parallel. Notice the change in the 5B12 and 1G09 profiles in UCP recovered from mice. NEMC1 samples and one MD sample in 1G09 were included as PCR controls. neg., negative PCR control.