Investigating within-host population diversity of Cryptosporidium parvum using BlooMine.

Investigating multiplicity-of-infection (MOI) in pathogen populations is central to understanding within-host evolutionary dynamics. In Cryptosporidium parvum, MOI may play a pivotal role in diversification due to the parasite's sexually recombinogenic life cycle, which occurs entirely within a single host. Subtyping of C. parvum typically relies on variation at short tandem repeat (STR) loci - such as that found in the 60kDa surface glycoprotein gene, Gp60 - but accurate profiling of these regions from next-generation sequencing (NGS) data remains technically challenging. Here, we apply BlooMine, an alignment-free, Bloom filter based tool that isolates STR containing reads via a novel pseudo-alignment strategy robust to structural and sequence variation. Using this approach, we analyse the full publicly available C. parvum Illumina dataset to quantify polyclonality at the Gp60 locus. After stringent artefact suppression, we detect strong evidence of in-host diversity across multiple continents and hosts. Cattle samples exhibited a 2.3-fold higher odds of harbouring multiple Gp60 subtypes compared to human samples, a difference that remained significant after adjustment for geography, sequencing depth, and subtype family. Allele co-occurrence analysis revealed closely related pairs likely arising from replication slippage, as well as mutually exclusive combinations suggestive of within-host competition or transmission structuring. Several subtypes traditionally considered host-specific were detected in unexpected host contexts, indicating greater host plasticity than previously assumed. Our results support a model in which STR-driven microevolution, recombination, and polyclonal infection jointly shape C. parvum population structure. We propose that MOI may act as a genetic crucible, facilitating subtype diversification within individual hosts. This study represents the largest genomic survey of Gp60 polyclonality to date and provides key insights into the evolutionary and epidemiological dynamics of C. parvum.