Metabarcoding reveals mixed Cryptosporidium and apicomplexan infections in New Zealand dairy cattle

Cryptosporidium species are significant enteric parasites of cattle with zoonotic potential. Molecular surveillance of bovine cryptosporidiosis typically relies on 18S rRNA PCR and Sanger sequencing, which may underestimate mixed infections and overlook broader apicomplexan co-infections within individual hosts. High-throughput metabarcoding can easily characterize within-sample species diversity and subtype heterogeneity. Here, we combined conventional 18S rRNA PCR, 18S metabarcoding and gp60 metabarcoding to characterize Cryptosporidium species composition and subtype diversity in faecal samples collected from dairy cattle aged 2–20 weeks in the Canterbury and Manawatu regions of New Zealand. Of 98 faecal samples screened, 45 (46%) were confirmed positive for Cryptosporidium by nested 18S rRNA PCR and Sanger sequencing, identifying C. parvum (33/45, 73.3%), C. bovis (8/45, 17.8%) and C. ryanae (4/45, 8.9%). All PCR-positive samples were subsequently analysed using 18S metabarcoding to investigate mixed infections and broader apicomplexan community composition. Samples identified as C. bovis were further characterized using gp60 metabarcoding. Illumina-based 18S metabarcoding revealed complex apicomplexan communities in Cryptosporidium -positive samples, including frequent co-detection of Eimeria species. Cryptosporidium bovis -positive samples identified multiple subtype families within the XXVI group with evidence of mixed subtype-family infections in individual animals. These findings highlight that metabarcoding provides substantially improved resolution for bovine Cryptosporidium surveillance and reveals infection complexity that is systematically missed by conventional Sanger-based workflows. This enhanced detection capability offers valuable insights for epidemiological studies and informs One Health strategies addressing bovine cryptosporidiosis and its zoonotic risks.