Development and Application of a gp60-Based Subtyping Tool for Cryptosporidium bovis

Abstract

Cryptosporidium bovis is a common enteric pathogen in bovine animals. The research on transmission characteristics of the pathogen is hampered by the lack of subtyping tools. In this study, we retrieve the nucleotide sequence of the 60 kDa glycoprotein (GP60) from the whole genome sequences of C. bovis we obtained previously and analyze its sequence characteristics. Despite a typical structure of the GP60 protein, the GP60 of C. bovis had only 19.3%–45.3% sequence identity to those of other Cryptosporidium species. On the basis of the gene sequence, a subtype typing tool was developed for C. bovis and used in the analysis of 486 C. bovis samples from dairy cattle, yaks, beef cattle, and water buffalos from China. Sixty-eight sequence types were identified from 260 subtyped samples, forming six subtype families, namely XXVIa to XXVIf. The mosaic sequence patterns among subtype families and the 121 potential recombination events identified among the sequences both suggest the occurrence of genetic recombination at the locus. No obvious host adaptation and geographic differences in the distribution of subtype families were observed. Most farms with more extensive sampling had more than one subtype family, and the dominant subtype families on a farm appeared to differ between pre- and post-weaned calves, indicating the likely occurrence of multiple episodes of C. bovis infections. There was an association between XXVId infection and occurrence of moderate diarrhea in dairy cattle. The subtyping tool developed and the data generated in the study might improve our knowledge of the genetic diversity and transmission of C. bovis.

An Update on Zoonotic Cryptosporidium Species and Genotypes in Humans

“…A gp60 typing system has been established for C. bovis based on WGS sequences. Sixty-eight subtypes in six subtype families (XXVIa to XXVIf) have been identified, with apparent genetic recombination among subtype families [ 50 ]. There have been several reports of C. bovis in humans ( Table 1 ).…”

“…A more complete understanding of the molecular epidemiology of Cryptosporidium and zoonotic transmission dynamics can be gained from WGS studies, which are also increasingly being used to identify gp60 loci in divergent species and genotypes [ 235 ]. Currently, WGS data has been generated from the following zoonotic species and genotypes; C. parvum , C. hominis , C. muris , C. meleagridis , C. ubiquitum , C. andersoni , C. tyzzeri , C. cuniculus , C. viatorum , C. felis , C. canis , C. bovis , C. xiaoi , Cryptosporidium skunk genotype and chipmunk genotype I [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 236 ]. Comparative WGS has identified a variety of invasion-associated proteins including mucin glycoproteins, insulinase-like proteases and MEDLE secretory proteins, which differ between species with narrow and broad host ranges and hence may play a role in host specificity [ 76 , 237 , 238 , 239 ].…”

“…For example, C. hominis subtype IfA19G1R5 has 19 TCA (A) repeats, 1 TCG repeat (G) repeat and 5 AAGAAGGCAAAGAAG repeats (R). For some divergent species and genotypes, including C. ubiquitum, C. felis, C. canis, C. ryanae, C. xiaoi, C. bovis and Cryptosporidium chipmunk genotype I and skunk genotype, whole-genome sequencing (WGS) has been required to identify the gp60 loci in order to develop typing systems [ 44 , 45 , 46 , 47 , 48 , 49 , 50 ]. The trinucleotide repeats (TCA, TCG, TCT) are absent from gp60 genes in C. ubiquitum, C. felis, C. canis, C. ryanae, C. bovis and C. xiaoi [ 44 , 46 , 47 , 48 , 49 , 50 ].…”

Age and episode‐associated occurrence of Cryptosporidium species and subtypes in a birth‐cohort of dairy calves

“…The species present was determined by sequence analysis of the PCR products. The C. parvum , C. bovis and C. ryanae were subtyped by PCR analysis of ∼830‐bp, ∼1300‐bp and ∼1000‐bp fragments of the gp60 gene, respectively (Feng et al., 2009; Yang et al., 2020; Wang et al., 2021). Each DNA preparation was analysed by PCR at each genetic locus at least twice.…”

Editorial for the Special Issue: Diagnosis, Epidemiology and Transmission Dynamics of Cryptosporidium spp. and Giardia duodenalis

“…The authors tested 355 C. xiaoi-positive samples from Chinese sheep and goats and found an extremely large intra-species genetic diversity, resulting in the identification of 12 (XXIIIa to XXIIIl) subtype families. This study complements and expands the available gp60 subtyping toolbox, including protocols adapted to Cryptosporidium species, such as C. hominis and C. parvum [23], C. fayeri [24], C. meleagridis [25], C. ubiquitum [26], C. viatorum [27], C. felis [22], C. ryanae [28], C. canis [29], and C. bovis [30].…”