Multilocus sequence typing of an emerging Cryptosporidium hominis subtype in the United States

Abstract

The United States has experienced a substantial increase in the reported incidence of cryptosporidiosis since 2005. Accompanying this is the emergence of a new subtype of Cryptosporidium hominis based on variation at the 60-kDa glycoprotein (gp60) locus, IaA28R4, which has become a frequently identified subtype in both sporadic and outbreak-related cases. In this study, using multilocus sequence typing (MLST) at eight genetic loci, we characterized 62 specimens of IaA28R4 and 33 specimens of three other gp60 subtypes of C. hominis from four U.S. states with increased cryptosporidiosis incidences during the summer of 2008. Extensive genetic heterogeneity was seen within the gp60 subtype IaA28R4, but specimens from Ohio and southwestern states formed two distinct subpopulations, suggesting that there were at least two origins of IaA28R4 within the United States. Discordance in typing results was observed between gp60 and other genetic markers, especially DZ-HRGP, and this discordance was largely the result of genetic recombination within the gp60 subtype IaA28R4. The results of population genetic analyses supported the presence of two subpopulations of IaA28R4 and the occurrence of genetic recombination within this gp60 subtype. Thus, the IaA28R4 subtype at gp60 is likely a fitness marker for C. hominis, and genetic recombination is potentially a driving force in the emergence of the virulent IaA28R4 subtype in the United States. A rapid evolution of IaA28R4 was indicated by the observation of multiple MLST subtypes of IaA28R4 within two large outbreaks that lasted for extended periods and involved multiple swimming pools.

FIG 1

Population substructure of 95 specimens of C. hominis from Ohio, Texas, New Mexico, and Arizona in the United States by Bayesian analyses of the MLST allelic data. Subpopulation patterns are shown for K values of 2 to 6 used in the analysis. Colored regions indicate major ancestral contributions. Mixed subtypes are indicated by the pattern of color combinations. Subtype identities of the specimens at the gp60 locus are shown above the color patterns, and the geographic origins of the specimens are shown below the color patterns. T1 and T2, specimens from an outbreak in two neighboring northeastern counties in Texas; T3, specimens from sporadic cases in a central Texas city; NM, specimens from sporadic cases (except for two IaA15R3 specimens) from New Mexico; AZ, specimens from an outbreak in Arizona; OH1 and OH2, specimens from two outbreaks in central Ohio. All specimens are from 2008, except for OH1, which represents two specimens from 2005.

FIG 2

Patterns of evolutionary descent among multilocus sequence typing (MLST) types of Cryptosporidium hominis, as revealed by eBURST analysis of allelic data. The numbers for each MLST type represent the sequence type at each of the eight MLST loci, in the following order: gp60, CP47, Mucin1, CP56, RPGR, MSC6-7, DZ-HRGP, and HSP70. Blue dot, primary founder; yellow dots, subgroup founders. The size of each dot is proportional to the number of specimens of the sequence type. The three MLST types of the gp60 IgA27 subtype did not cluster in this diagram. See Fig. 1 legend for the origins of the specimens.