BACKGROUND: Cryptosporidium parvum is a zoonotic pathogen worldwide. Extensive genetic diversity and complex population structures exist in C. parvum in different geographical regions and hosts. Unlike the IIa subtype family, which is responsible for most zoonotic C. parvum infections in industrialized countries, IId is identified as the dominant subtype family in farm animals, rodents and humans in China. Thus far, the population genetic characteristics of IId subtypes in calves in China are not clear. METHODS: In the present study, 46 C. parvum isolates from dairy and beef cattle in six provinces and regions in China were characterized using sequence analysis of eight genetic loci, including msc6-7, rpgr, msc6-5, dz-hrgp, chom3t, hsp70, mucin1 and gp60. They belonged to three IId subtypes in the gp60 gene, including IIdA20G1 (n = 17), IIdA19G1 (n = 24) and IIdA15G1 (n = 5). The data generated were analyzed for population genetic structures of C. parvum using DnaSP and LIAN and subpopulation structures using STRUCTURE, RAxML, Arlequin, GENALEX and Network. RESULTS: Seventeen multilocus genotypes were identified. The results of linkage disequilibrium analysis indicated the presence of an epidemic genetic structure in the C. parvum IId population. When isolates of various geographical areas were treated as individual subpopulations, maximum likelihood inference of phylogeny, pairwise genetic distance analysis, substructure analysis, principal components analysis and network analysis all provided evidence for geographical segregation of subpopulations in Heilongjiang, Hebei and Xinjiang. In contrast, isolates from Guangdong, Shanghai and Jiangsu were genetically similar to each other. CONCLUSIONS: Data from the multilocus analysis have revealed a much higher genetic diversity of C. parvum than gp60 sequence analysis. Despite an epidemic population structure, there is an apparent geographical segregation in C. parvum subpopulations within China.

Center for Emerging and Zoonotic Diseases College of Veterinary Medicine South China Agricultural University Guangzhou Guangdong 510642 China

College of Animal Science and Veterinary Medicine Henan Agricultural University Zhengzhou 450002 China

Guangdong Laboratory for Lingnan Modern Agriculture Guangzhou 510642 China

Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic České Budějovice 37005 Czech Republic

See more in PubMed

Checkley W, White AC, Jaganath D, Arrowood MJ, Chalmers RM, Chen X, Fayer R, et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis. 2015;15:85–94. doi: 10.1016/S1473-3099(14)70772-8. PubMed DOI PMC

Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol. 2018;34:997–1011. doi: 10.1016/j.pt.2018.07.009. PubMed DOI

Xiao L, Feng Y. Molecular epidemiologic tools for waterborne pathogens Cryptosporidium sp and Giardia duodenalis. Food Waterborne Parasitol. 2017;8–9:14–32. doi: 10.1016/j.fawpar.2017.09.002. PubMed DOI PMC

Nader JL, Mathers TC, Ward BJ, Pachebat JA, Swain MT, Robinson G, et al. Evolutionary genomics of anthroponosis in Cryptosporidium. Nat Microbiol. 2019;4:826–836. doi: 10.1038/s41564-019-0377-x. PubMed DOI

Ryan U, Fayer R, Xiao L. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology. 2014;141:1667–1685. doi: 10.1017/S0031182014001085. PubMed DOI

Xiao L. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol. 2010;124:80–89. doi: 10.1016/j.exppara.2009.03.018. PubMed DOI

Feng Y, Xiao L. Molecular epidemiology of cryptosporidiosis in China. Front Microbiol. 2017;8:1701–1711. doi: 10.3389/fmicb.2017.01701. PubMed DOI PMC

Li N, Wang R, Cai M, Jiang W, Feng Y, Xiao L. Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China. Int J Parasitol. 2019;49:569–577. doi: 10.1016/j.ijpara.2019.02.006. PubMed DOI PMC

Cui Z, Wang R, Huang J, Wang H, Zhao J, Luo N, et al. Cryptosporidiosis caused by Cryptosporidium parvum subtype IIdA15G1 at a dairy farm in northwestern China. Parasit Vectors. 2014;7:529. doi: 10.1186/s13071-014-0529-z. PubMed DOI PMC

Widmer G, Lee Y. Comparison of single- and multilocus genetic diversity in the protozoan parasites Cryptosporidium parvum and C. hominis. Appl Environ Microb. 2010;76:6639–6644. doi: 10.1128/AEM.01268-10. PubMed DOI PMC

Chalmers RM, PérezCordón G, Cacció SM, Klotz C, Robertson LJ. Participants of the Cryptosporidium genotyping workshop (EURO-FBP) Cryptosporidium genotyping in Europe: the current status and processes for a harmonised multi-locus genotyping scheme. Exp Parasitol. 2018;191:25–30. doi: 10.1016/j.exppara.2018.06.004. PubMed DOI

Mallon ME, MacLeod A, Wastling JM, Smith H, Tait A. Multilocus genotyping of Cryptosporidium parvum Type 2: population genetics and sub-structuring. Infect Genet Evol. 2003;3:207–218. doi: 10.1016/S1567-1348(03)00089-3. PubMed DOI

Cacció SM, de Waele V, Widmer G. Geographical segregation of Cryptosporidium parvum multilocus genotypes in Europe. Infect Genet Evol. 2015;31:245–249. doi: 10.1016/j.meegid.2015.02.008. PubMed DOI

De Waele V, Van den Broeck F, Huyse T, McGrath G, Higgins I, Speybroeck N, et al. Panmictic structure of the Cryptosporidium parvum population in Irish calves: influence of prevalence and host movement. Appl Environ Microbiol. 2013;79:2534–2541. doi: 10.1128/AEM.03613-12. PubMed DOI PMC

Drumo R, Widmer G, Morrison LJ, Tait A, Grelloni V, D’Avino N, et al. Evidence of host-associated populations of Cryptosporidium parvum in Italy. Appl Environ Microbiol. 2012;78:3523–3529. doi: 10.1128/AEM.07686-11. PubMed DOI PMC

Feng Y, Torres E, Li N, Wang L, Bowman D, Xiao L. Population genetic characterisation of dominant Cryptosporidium parvum subtype IIaA15G2R1. Int J Parasitol. 2013;43:1141–1147. doi: 10.1016/j.ijpara.2013.09.002. PubMed DOI

Herges GR, Widmer G, Clark ME, Khan E, Giddings CW, Brewer M, et al. Evidence that Cryptosporidium parvum populations are panmictic and unstructured in the Upper Midwest of the United States. Appl Environ Microbiol. 2012;78:8096–8101. doi: 10.1128/AEM.02105-12. PubMed DOI PMC

Mallon M, MacLeod A, Wastling J, Smith H, Reilly B, Tait A. Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum. J Mol Evol. 2003;56:407–417. doi: 10.1007/s00239-002-2412-3. PubMed DOI

Morrison LJ, Mallon ME, Smith HV, MacLeod A, Xiao L, Tait A. The population structure of the Cryptosporidium parvum population in Scotland: a complex picture. Infect Genet Evol. 2008;8:121–129. doi: 10.1016/j.meegid.2007.10.010. PubMed DOI PMC

Ramo A, Quílez J, Monteagudo L, Del Cacho E, Sánchez-Acedo C. Intra-species diversity and panmictic structure of Cryptosporidium parvum populations in cattle farms in northern Spain. PloS ONE. 2016;11:e0148811. doi: 10.1371/journal.pone.0148811. PubMed DOI PMC

Tanriverdi S, Markovics A, Arslan MO, Itik A, Shkap V, Widmer G. Emergence of distinct genotypes of Cryptosporidium parvum in structured host populations. Appl Environ Microbiol. 2006;72:2507–2513. doi: 10.1128/AEM.72.4.2507-2513.2006. PubMed DOI PMC

Ramo A, Monteagudo LV, Del Cacho E, Sánchez-Acedo C, Quílez J. Intra-species genetic diversity and clonal structure of Cryptosporidium parvum in sheep farms in a confined geographical area in northeastern Spain. PloS One. 2016;11:e0155336. doi: 10.1371/journal.pone.0155336. PubMed DOI PMC

Feng Y, Gong X, Zhu K, Li N, Yu Z, Guo Y, et al. Prevalence and genotypic identification of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in pre-weaned dairy calves in Guangdong, China. Parasit Vectors. 2019;12:41. PubMed PMC

Cai M, Guo Y, Pan B, Li N, Wang X, Tang C, et al. Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai China. Vet Parasitol. 2017;241:14–19. doi: 10.1016/j.vetpar.2017.05.005. PubMed DOI

Wang R, Zhang L, Axen C, Bjorkman C, Jian F, Amer S, et al. Cryptosporidium parvum IId family: clonal population and dispersal from western Asia to other geographical regions. Sci Rep. 2014;4:4208. doi: 10.1038/srep04208. PubMed DOI PMC

Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34:3299–3302. doi: 10.1093/molbev/msx248. PubMed DOI

Haubold B, Hudson RR. LIAN 3.0: detecting linkage disequilibrium in multilocus data. Linkage analysis. Bioinformatics. 2000;16:847–848. doi: 10.1093/bioinformatics/16.9.847. PubMed DOI

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. doi: 10.1093/bioinformatics/btu033. PubMed DOI PMC

Hubisz MJ, Falush D, Stephens M, Pritchard JK. Inferring weak population structure with the assistance of sample group information. Mol Ecol Resour. 2009;9:1322–1332. doi: 10.1111/j.1755-0998.2009.02591.x. PubMed DOI PMC

Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics. 2012;28:2537–2539. doi: 10.1093/bioinformatics/bts460. PubMed DOI PMC

Bandelt HJ, Forster P, Rohl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999;16:37–48. doi: 10.1093/oxfordjournals.molbev.a026036. PubMed DOI

Wang R, Zhao G, Gong Y, Zhang L. Advances and perspectives on the epidemiology of bovine Cryptosporidium in China in the past 30 years. Front Microbiol. 2017;8:1823. doi: 10.3389/fmicb.2017.01823. PubMed DOI PMC

Tanriverdi S, Grinberg A, Chalmers RM, Hunter PR, Petrovic Z, Akiyoshi DE, et al. Inferences about the global population structures of Cryptosporidium parvum and Cryptosporidium hominis. Appl Environ Microbiol. 2008;74:7227–7234. doi: 10.1128/AEM.01576-08. PubMed DOI PMC

Quílez J, Vergara-Castiblanco C, Monteagudo L, Del Cacho E, Sánchez-Acedo C. Multilocus fragment typing and genetic structure of Cryptosporidium parvum isolates from diarrheic preweaned calves in Spain. Appl Environ Microbiol. 2011;77:7779–7786. doi: 10.1128/AEM.00751-11. PubMed DOI PMC

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference