Several divergent Cryptosporidium parvum subtypes have emerged in people in recent years, but their infectivity, pathogenicity, and genetic characteristics are unclear. In the present study, IFN-γ knockout C57BL/6 (GKO) mice were infected with the novel IIoA15G1 and IIpA11 subtypes of C. parvum and the common IIaA17G2R1 subtype. The genomes of these isolates were sequenced and compared with each other. Further gene tagging and deletion were performed on the most polymorphic virulence-associated cgd8_5420 gene encoding a hypothetical protein using the CRISPR/Cas9 technology. IIpA11 and IIoA15G1 were highly infectious in GKO mice, with an ID50 of 2.4 and 3.6 oocysts, respectively. The duration of oocyst shedding for IIpA11 (>58.0 ± 1.4 d) and IIoA15G1 (>57.5 ± 0.9 d) was significantly longer than for IIaA17G2R1 (5.5 ± 0.9 d; p < 0.001). One of the mice infected with IIpA11 died on day 33 post infection. The genomes of IIaA17G2R1, IIoA15G1, and IIpA11 had 203, 46839, and 47,122 single nucleotide polymorphisms, respectively, compared to C. parvum IOWA II. In contrast, only 3,361 nucleotide differences were found between IIoA15G1 and IIpA11, with several genes encoding invasion-associated mucin glycoproteins and cgd8_5420 encoding a secretory protein being highly polymorphic. The latter is mainly expressed in trophozoites, merozoites, and macrogametes. Deletion of this gene reduced the intensity of IIpA11 infection and increased the survival of infected mice. Therefore, the emerging IIoA15G1 and IIpA11 subtypes have divergent genomes compared to common IIa subtypes and are highly infectious and pathogenic in GKO mice. Several secretory proteins, including a variant protein encoded by the subtelomeric cgd8_5420 gene, are associated with differences in virulence between the two subtypes.

Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation Fuyang Normal University Fuyang Anhui China

Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic České Budějovice Czech Republic

State Key Laboratory for Animal Disease Control and Prevention Center for Emerging and Zoonotic Diseases College of Veterinary Medicine South China Agricultural University Guangzhou Guangdong China

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BioProject
PRJNA1173948, PRJNA1173943, PRJNA1173945

SRA
SRR31044660, SRR31034905, SRR31035168

GENBANK
JBIQPL000000000, JBIQPK000000000, JBIPKD000000000