Multilocus Fragment Typing and Genetic Structure of Cryptosporidium parvum Isolates from Diarrheic Preweaned Calves in Spain

Abstract

A collection of 140 Cryptosporidium parvum isolates previously analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequence analyses of the small-subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes was further characterized by multilocus fragment typing of six minisatellite (MSB and MS5) and microsatellite (ML1, ML2, TP14, and 5B12) loci. Isolates were collected from diarrheic preweaned calves originating from 61 dairy cattle farms in northern Spain. A capillary electrophoresis-based tool combining three different fluorescent tags was used to analyze all six satellites in one capillary. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis of a selection of isolates for every allele. Size discrepancies at all but the 5B12 locus were found for those isolates that were typed by both techniques, although identical size differences were reported for every allele within each locus. A total of eight alleles were seen at the ML2 marker, which contributed the most to the discriminatory power of the multilocus approach. Multilocus fragment typing clearly improved the discriminatory power of GP60 sequencing, since a total of 59 multilocus subtypes were identified based on the combination of alleles at the six satellite loci, in contrast to the 7 GP60 subtypes previously reported. The majority of farms (38) displayed a unique multilocus subtype, and individual isolates with mixed multilocus subtypes were seen at 22 farms. Bayesian structure analysis based on combined data for both satellite and GP60 loci suggested the presence of two major clusters among the C. parvum isolates from cattle farms in this geographical area.

Evidence of Host-Associated Populations of Cryptosporidium parvum in Italy

“…Ideally, an interlaboratory ring trial should be arranged to identify those markers. Results from the present study and previously published studies (see, e.g., reference 16) showed that some markers (e.g., GP60 and MM19) are particularly polymorphic in C. parvum, whereas others (e.g., ML1) have a low level of polymorphism (12,13,18) and could be omitted. There are also technical issues to consider, such as the discrimination of alleles having identical size but different nucleotide sequences and the ability of various genotyping methods to detect minority populations in mixed infections (18).…”

“…Extensive diversity has been reported previously, such as that observed in 23 isolates from Serbia, which were all unique MLGs, and in isolates from Turkey, where 9 different MLGs were found in 11 isolates (26); lower levels appear to characterize isolates from Spain (59 MLGs in 137 isolates) (18) and Scotland (95 MLGs in 297 isolates) (16).…”

“…Differences can also be explained by a reduced frequency of recombination between isolates in countries where modern husbandry practices restrict herd-to-herd transmission. Under such circumstances, specific MLGs may be restricted to individual farms, as observed in Israel and Spain (18,24) and in this study. However, animal movement can counteract this effect, as reported by Morrison et al (16), who have clearly shown a strong correlation between parasite genetic diversity in defined areas and animal movement within the country.…”

“…A recent study from Spain (18) showed that some alleles commonly found in lambs and goat kids are rarely found in calves from the same region, indicating a similar degree of host association. Therefore, the finding that different subpopulations of the parasite are associated with different livestock species appears to be valid for goats but not for sheep.…”

“…The level of mixed infection in C. parvum populations varies considerably in published data (16,18,24), from no detection in New Zealand (19 human and 7 calf isolates) to 8.2% in Israel (59 calf isolates, 1 goat isolate, and 1 horse isolate), 19% in Uganda (36 human isolates), 20% in Spain (140 calf isolates), 30% in Serbia (23 calf isolates), 53% in Turkey (15 calf isolates), and 10 to 37% in four regions of Scotland (65 human, 211 calf, and 9 sheep isolates). These differences may in part reflect differential sensitivity of either the genotyping method or sets of markers for detecting minority populations or sampling effects (in terms of size and host representation).…”