High pathogenicity of Cryptosporidium parvum IId subtypes in dairy calves

Abstract

Zoonotic cryptosporidiosis is caused primarily by Cryptosporidium parvum. Within C. parvum, the IIa and IId zoonotic subtype families are the most prevalent. The IId subtype family has emerged in China in recent years, causing outbreaks of cryptosporidiosis in dairy calves. The majority of infection studies have been conducted with IIa subtypes, while the pathogenicity of IId subtypes remains poorly understood. In this study, two IId isolates (IIdA20G1-HLJ and IIdA20G1-HB) from dairy farms in China were used to infect neonatal dairy calves, with a IIa isolate (IIaA17G2R1-Waterborne) from the USA employed as a control. The present study investigated the clinical, parasitological, and pathological characteristics of infected calves. The results demonstrated significantly greater intensity and duration of oocyst shedding in IId-infected calves than in control calves. In addition, the IIdA20G1-HLJ isolate induced peak oocyst shedding of 4.3×10⁷ oocysts per gram of feces (OPG) in calves, with oocyst shedding over 10⁶ OPGs at 5–9 d post-infection. In contrast, the IIaA17G2R1-Waterbrone isolate induced a lower peak oocyst shedding with 8.7×10⁶ OPGs, and oocyst shedding with over 10⁶ OPGs occurred at 2–4 d post-infection. Furthermore, calves infected with the two IId isolates presented more severe clinical signs and 20–25% mortality. Calves recovered from the primary infection with the IIa or IId subtype were resistant to a secondary challenge with the heterologous subtype, suggesting the existence of cross-protection between the IIa and IId subtypes. The present data demonstrate the high pathogenicity of C. parvum IId subtypes in China and the potential cross-protective immunity between the IIa and IId subtypes in calves.

Keywords

Cryptosporidium / Cryptosporidiosis / Subtype / Calves / Pathogenicity

References

[1] AlvesM, XiaoL, SulaimanI, LalAA, MatosO, AntunesF. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. Journal of Clinical Microbiology, 2003, 41: 2744-2747.

[2] ArrowoodMJ, DonaldsonK. Improved purification methods for calf-derived Cryptosporidium parvum oocysts using discontinuous sucrose and cesium chloride gradients. The Journal of Eukaryotic Microbiology, 1996, 4389S.

[3] AudebertC, BonardiF, CabocheS, GuyotK, TouzetH, MerlinS, GantoisN, CreusyC, MeloniD, MourayA, et al.. Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates. Scientific Reports, 2020, 107316.

[4] CheckleyW, WhiteACJr., JaganathD, ArrowoodMJ, ChalmersRM, ChenXM, FayerR, GriffithsJK, GuerrantRL, et al.. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. The Lancet Infectious Diseases, 2015, 15: 85-94.

[5] CollaboratorsGBDDD. Global, regional, and national age-sex-specific burden of diarrheal diseases, their risk factors, and etiologies, 1990–2021, for 204 countries and territories: A systematic analysis for the Global Burden of Disease Study 2021. The Lancet Infectious Diseases, 2024, S1473–3099: 00691-00691.

[6] FengY, RyanUM, XiaoL. Genetic diversity and population structure of Cryptosporidium. Trends in Parasitology, 2018, 34: 997-1011.

[7] GamsjagerL, CironeKM, SchluesselS, CampsallM, HerikA, LahiriP, YoungD, DufourA, SapountzisP, OtaniS, et al.. Host innate immune responses and microbiome profile of neonatal calves challenged with Cryptosporidium parvum and the effect of bovine colostrum supplementation. Frontiers in Cellular and Infection Microbiology, 2023, 131165312.

[8] GilchristCA, CampoJJ, PabloJV, MaJZ, TengA, OberaiA, ShandlingAD, AlamM, KabirM, et al.. Specific Cryptosporidium antigens associate with reinfection immunity and protection from cryptosporidiosis. Journal of Clinical Investigation, 2023.

[9] GuoY, LiN, RyanU, FengY, XiaoL. Small ruminants and zoonotic cryptosporidiosis. Parasitology Research, 2021, 120: 4189-4198.

[10] GuoY, RyanU, FengY, XiaoL. Emergence of zoonotic Cryptosporidium parvum in China. Trends in Parasitology, 2022, 38: 335-343.

[11] HeW, SunL, HouT, YangZ, YangF, ZhangS, WangT, WangX, LiN, GuoY, et al.. SKSR1 identified as key virulence factor in Cryptosporidium by genetic crossing. Nature Communications, 2025, 164694.

[12] HuS, WanM, HuangW, WangW, LiangR, SuD, LiN, XiaoL, FengY, GuoY. Age and episode-associated occurrence of Cryptosporidium species and subtypes in a birth-cohort of dairy calves. Transboundary and Emerging Diseases, 2022.

[13] JiaR, HuangW, HuangN, YuZ, LiN, XiaoL, FengY, GuoY. High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China. PLoS Neglected Tropical Diseases, 2022, 16. e0010714

[14] JiangJ, AlderisioKA, SinghA, XiaoL. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Applied and Environmental Microbiology, 2005, 71: 1135-1141.

[15] LiN, NeumannNF, RueckerN, AlderisioKA, SturbaumGD, VillegasEN, ChalmersR, MonisP, FengY, XiaoL. Development and evaluation of three real-time PCR assays for genotyping and source tracking Cryptosporidium spp. in water. Applied and Environmental Microbiology, 2015, 81: 5845-5854.

[16] LiN, WangR, CaiM, JiangW, FengY, XiaoL. Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China. International Journal for Parasitology, 2019, 49: 569-577.

[17] LiN, ZhaoWT, SongSJ, YeHJ, ChuWL, GuoYQ, FengYY, XiaoLH. Diarrhea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in preweaned dairy calves. Transboundary and Emerging Diseases, 2022, 69: E1606-E1617.

[18] MeadJR. Prospects for immunotherapy and vaccines against Cryptosporidium. Human Vaccines & Immunotherapeutics, 2014, 10: 1505-1513.

[19] PerrymanLE, KapilSJ, JonesML, HuntEL. Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein. Vaccine, 1999, 17: 2142-2149.

[20] RiggsMW, SchaeferDA. Calf clinical model of cryptosporidiosis for efficacy evaluation of therapeutics. Methods in Molecular Biology, 2020, 2052: 253-282.

[21] RyanU, ZahediA, FengY, XiaoL. An update on zoonotic Cryptosporidium species and genotypes in humans. Animals, 2021.

[22] SantinM. Cryptosporidium and Giardia in ruminants. Veterinary Clinics of North America Food Animal Practice, 2020, 36: 223-238.

[23] Sayed, F.G., Hamza, A.I., Galal, L.A., Sayed, D.M., Gaber, M., 2016. Virulence of geographically different Cryptosporidium parvum isolates in experimental animal model. Annals of parasitology 62: 221–232. https://doi.org/10.17420/ap6203.56

[24] SchaeferDA, BetzerDP, SmithKD, MillmanZG, MichalskiHC, MenchacaSE, ZambriskiJA, OjoKK, HulversonMA, ArnoldSL, et al.. Novel bumped kinase inhibitors are safe and effective therapeutics in the calf clinical model for cryptosporidiosis. The Journal of Infectious Diseases, 2016, 214: 1856-1864.

[25] SheoranA, WiffinA, WidmerG, SinghP, TziporiS. Infection with Cryptosporidium hominis provides incomplete protection of the host against Cryptosporidium parvum. Journal of Infectious Diseases, 2012, 205: 1019-1023.

[26] SteinerKL, KabirM, HossainB, GilchristCA, MaJZ, AhmedT, FaruqueAG, HaqueR, PetriWA. Delayed time to cryptosporidiosis in Bangladeshi children is associated with greater fecal IgA against two sporozoite-expressed antigens. American Journal of Tropical Medicine and Hygiene, 2021, 104: 229-232.

[27] Timmermans, M., Hubers, W., Schroer, D., Gevers, K., Segers, R.P.A.M., Niessen, R., van Roosmalen, M.H., 2024. The first commercially approved efficacious Cryptosporidium vaccine protecting new-born calves from severe diarrhea. Veterinary Vaccine 3. https://doi.org/10.1016/j.vetvac.2024.100054

[28] Wang, T., Guo, Y., Roellig, D.M., Li, N., Santin, M., Lombard, J., Kvac, M., Naguib, D., Zhang, Z., Feng, Y., Xiao, L., 2022. Sympatric recombination in zoonotic Cryptosporidium leads to emergence of populations with modified host preference. Mol Biol Evol 39: msac150. https://doi.org/10.1093/molbev/msac150

[29] XiaoL, FengY. Molecular epidemiologic tools for waterborne pathogens Cryptosporidium spp. and Giardia duodenalis. Food and Waterborne Parasitology, 2017, 8–9: 14-32.

[30] XiaoL, EscalanteL, YangC, SulaimanI, EscalanteAA, MontaliRJ, FayerR, LalAA. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Applied and Environmental Microbiology, 1999, 65: 1578-1583.

[31] XiaoL, MorganUM, LimorJ, EscalanteA, ArrowoodM, ShulawW, ThompsonRC, FayerR, LalAA. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Applied and Environmental Microbiology, 1999, 65: 3386-3391.

[32] ZambriskiJA, NydamDV, WilcoxZJ, BowmanDD, MohammedHO, LiottaJL. Cryptosporidium parvum: Determination of ID(5)(0) and the dose-response relationship in experimentally challenged dairy calves. Veterinary Parasitology, 2013, 197: 104-112.

[33] ZhangZJ, SuDL, MengXA, LiangRB, WangWJ, LiN, GuoYQ, GuoAZ, LiSJ, ZhaoZY, et al.. Cryptosporidiosis outbreak caused by Cryptosporidium parvum subtype IIdA20G1 in neonatal calves. Transboundary and Emerging Diseases, 2022, 69: 278-285.

Funding

National Natural Science Foundation of China(32030109)

Guangdong Major Project of Basic and Applied Basic Research(2020B0301030007)

Postdoctoral Research Foundation of China(2023TQ0122)

Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center)(D20008)

Guangzhou Basic and Applied Basic Research Foundation(2025A04J4556)

National Key Clinical Specialty Discipline Construction Program of China(2023B10564003)

RIGHTS & PERMISSIONS

The Author(s)