Skip to main content
Log in

Tracking Cryptosporidium in urban wastewater treatment plants in a cold region: Occurrence, species and infectivity

  • Research Article
  • Published:
Frontiers of Environmental Science & Engineering Aims and scope Submit manuscript

Abstract

This study investigated the occurrence, species, infectivity and removal efficiency of Cryptosporidium spp. across typical wastewater treatment train. Samples from different process units were collected seasonally and synchronously from four wastewater treatment plants (WWTPs) in Northeastern China. Live Cryptosporidium oocysts were identified in most samples from both influent (97.50%) and effluent (90.00%) wastewaters of the four WWTPs, at an average density of 26.34 and 4.15 oocysts/L, respectively. The overall removal efficiency was 84.25%, and oocysts were mainly removed (62.01%) by the modified secondary sedimentation process. Ten Cryptosporidium species were identified in the effluent samples. C. andersoni, C. bovis, and C. ryanae were the three most prevalent species. Oocyst viability assays indicated no reduction of excystation rate during the primary and secondary wastewater treatments (varied in the range of 63.08%–68.50%), but the excystation rate declined to 52.21% in the effluent after disinfection. Notably, the Cryptosporidium oocysts showed higher infection intensity in the cold season (winter and spring) than that in summer and autumn. The influences of environmental temperature on virulence factors of Cryptosporidium were further examined. It was observed that more extracellular secretory proteins were bound on the oocyst surface and several virulence genes were expressed relatively strongly at low temperatures, both of which could facilitate oocyst adhesion, invasion, and host immune evasion. This research is of considerable interest since it serves as an important step towards more accurate panoramic recognition of Cryptosporidium risk reduction in WWTPs, and especially highlights the potential health risk associated with Cryptosporidium in cold regions/seasons.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Adeyemo F E, Singh G, Reddy P, Bux F, Stenström T A (2019). Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater. PLoS One, 14(5): e0216040

    Article  Google Scholar 

  • Andreoli F C, Sabogal-Paz L P (2019). Coagulation, flocculation, dissolved air flotation and filtration in the removal of Giardia spp. and Cryptosporidium spp. from water supply. Environmental Technology, 40(5): 654–663

    Article  CAS  Google Scholar 

  • Arrowood M J, Sterling C R (1987). Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. Journal of Parasitology, 73(2): 314–319

    Article  CAS  Google Scholar 

  • Barudin M A, Isa M L M, Yusof A M (2018). Signal peptide sequence analysis of selected protein sequences from Cryptosporidium parvum. Trends in Bioinformatics, 11(1): 33–43

    Article  CAS  Google Scholar 

  • Bouzid M, Hunter P R, Chalmers R M, Tyler K M (2013). Cryptosporidium pathogenicity and virulence. Clinical Microbiology Reviews, 26(1): 115–134

    Article  CAS  Google Scholar 

  • Bradford S A, Kim H, Headd B, Torkzaban S (2016). Evaluating the transport of Bacillus subtilis spores as a potential surrogate for Cryptosporidium parvum oocysts. Environmental Science & Technology, 50(3): 1295–1303

    Article  CAS  Google Scholar 

  • Cama V A, Ross J M, Crawford S, Kawai V, Chavez-Valdez R, Vargas D, Vivar A, Ticona E, Navincopa M, Williamson J, Ortega Y, Gilman R H, Bern C, Xiao L (2007). Differences in clinical manifestations among Cryptosporidium species and subtypes in HIV-infected persons. The Journal of Infectious Diseases, 196(5): 684–691

    Article  Google Scholar 

  • Castro-Hermida J A, García-Presedo I, Almeida A, González-Warleta M, Da Costa J M, Mezo M (2009). Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: A health risk for humans and animals. Water Research, 43(17): 4133–4142

    Article  CAS  Google Scholar 

  • Chauret C P, Radziminski C Z, Lepuil M, Creason R, Andrews R C (2001). Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators. Applied and Environmental Microbiology, 67(7): 2993–3001

    Article  CAS  Google Scholar 

  • Dreelin E A, Ives R L, Molloy S, Rose J B (2014). Cryptosporidium and Giardia in surface water: A case study from Michigan, USA to inform management of rural water systems. International Journal of Environmental Research and Public Health, 11(10): 10480–10503

    Article  Google Scholar 

  • Edzwald J K, Tobiason J E (2002). Fate and removal of Cryptosporidium in a dissolved air flotation water plant with and without recycle of waste filter backwash water. Water Science and Technology: Water Supply, 2(2): 85–90

    Google Scholar 

  • Falohun O O, Ayinmode A B, Adejinmi J O (2021). Molecular characterisation of Cryptosporidium isolates from rivers, water treatment plants and abattoirs in Ibadan, Nigeria. Comparative Immunology, Microbiology and Infectious Diseases, 74: 101577

    Article  CAS  Google Scholar 

  • Fan W, Yang X, Wang Y, Huo M (2020). Loopholes in the current reclaimed water quality standards for clogging control during aquifer storage and recovery in China. Water Cycle, 1: 13–18

    Article  Google Scholar 

  • Farrell C, Hassard F, Jefferson B, Leziart T, Nocker A, Jarvis P (2018). Turbidity composition and the relationship with microbial attachment and UV inactivation efficacy. Science of the Total Environment, 624: 638–647

    Article  CAS  Google Scholar 

  • Fayer R, Orlandi P, Perdue M L (2009). Virulence factor activity relationships for hepatitis E and Cryptosporidium. Journal of Water and Health, 7(S1 Suppl 1): S55–S63

    Article  Google Scholar 

  • Foster T J, Geoghegan J A, Ganesh V K, Höök M (2014). Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nature Reviews. Microbiology, 12(1): 49–62

    CAS  Google Scholar 

  • Fu C Y, Xie X, Huang J J, Zhang T, Wu Q Y, Chen J N, Hu H Y (2010). Monitoring and evaluation of removal of pathogens at municipal wastewater treatment plants. Water Science and Technology, 61(6): 1589–1599

    Article  CAS  Google Scholar 

  • Galván A L, Magnet A, Izquierdo F, Fernández Vadillo C, Peralta R H, Angulo S, Fenoy S, del Aguila C (2014). A year-long study of Cryptosporidium species and subtypes in recreational, drinking and wastewater from the central area of Spain. Science of the Total Environment, 468–469(468–469): 368–375

    Article  Google Scholar 

  • Gharpure R, Perez A, Miller A D, Wikswo M E, Silver R, Hlavsa M C (2019). Cryptosporidiosis outbreaks- United States, 2009–2017. Morbidity and Mortality Weekly Report, 68(25): 568–572

    Article  Google Scholar 

  • Hamilton K A, Waso M, Reyneke B, Saeidi N, Levine A, Lalancette C, Besner M C, Khan W, Ahmed W (2018). Cryptosporidium and Giardia in wastewater and surface water environments. Journal of Environmental Quality, 47(5): 1006–1023

    Article  CAS  Google Scholar 

  • Haramoto E, Kitajima M, Kishida N, Katayama H, Asami M, Akiba M (2012). Occurrence of viruses and protozoa in drinking water sources of Japan and their relationship to indicator microorganisms. Food and Environmental Virology, 4(3): 93–101

    Article  Google Scholar 

  • Hatam-Nahavandi K, Mohebali M, Mahvi A, Keshavarz H, Khanaliha K, Tarighi F, Molaei-Rad M, Rezaeian T, Charehdar S, Salimi M, Farnia S, Rezaeian M (2015). Evaluation of Cryptosporidium oocyst and Giardia cyst removal efficiency from urban and slaughter house wastewater treatment plants and assessment of cyst viability in wastewater effluent samples from Tehran, Iran. Journal of Water Reuse and Desalination, 5(3): 372–390

    Article  CAS  Google Scholar 

  • Headd B, Bradford S A (2016). Use of aerobic spores as a surrogate for Cryptosporidium oocysts in drinking water supplies. Water Research, 90: 185–202

    Article  CAS  Google Scholar 

  • Huang C, Hu Y, Wang L, Wang Y, Li N, Guo Y, Feng Y, Xiao L (2017). Environmental transport of emerging human-pathogenic Cryptosporidium species and subtypes through combined sewer overflow and wastewater. Applied and Environmental Microbiology, 83(16): e00682–e17

    Article  CAS  Google Scholar 

  • Jenkins M B, Eaglesham B S, Anthony L C, Kachlany S C, Bowman D D, Ghiorse W C (2010). Significance of wall structure, macro-molecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts. Applied and Environmental Microbiology, 76(6): 1926–1934

    Article  CAS  Google Scholar 

  • Kihara T, Ito J, Miyake J (2013). Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy. PLoS One, 8(11): e82382

    Article  Google Scholar 

  • King B, Fanok S, Phillips R, Lau M, van den Akker B, Monis P (2017). Cryptosporidium attenuation across the wastewater treatment train: recycled water fit for purpose. Applied and Environmental Microbiology, 83(5): e03068–e16

    Article  Google Scholar 

  • Kitajima M, Haramoto E, Iker B C, Gerba C P (2014). Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona. Science of the Total Environment, 484: 129–136

    Article  CAS  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7): 1870–1874

    Article  CAS  Google Scholar 

  • Lages M A, Balado M, Lemos M L (2019). The expression of virulence factors in Vibrio anguillarum is dually regulated by iron levels and temperature. Frontiers in Microbiology, 10: 2335

    Article  Google Scholar 

  • Lam O, Wheeler J, Tang C M (2014). Thermal control of virulence factors in bacteria: A hot topic. Virulence, 5(8): 852–862

    Article  Google Scholar 

  • Lapen D R, Schmidt P J, Thomas J L, Edge T A, Flemming C, Keithlin J, Neumann N, Pollari F, Ruecker N, Simhon A, Topp E, Wilkes G, Pintar K D M (2016). Towards a more accurate quantitative assessment of seasonal Cryptosporidium infection risks in surface waters using species and genotype information. Water Research, 105: 625–637

    Article  CAS  Google Scholar 

  • Li N, Xiao L, Wang L, Zhao S, Zhao X, Duan L, Guo M, Liu L, Feng Y (2012). Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Neglected Tropical Diseases, 6(9): e1809

    Article  Google Scholar 

  • Li X, Atwill E R, Dunbar L A, Jones T, Hook J, Tate K W (2005). Seasonal temperature fluctuations induces rapid inactivation of Cryptosporidium parvum. Environmental Science & Technology, 39(12): 4484–4489

    Article  CAS  Google Scholar 

  • Li X, Brasseur P, Agnamey P, Ballet J J, Clemenceau C (2004). Time and temperature effects on the viability and infectivity of Cryptosporidium parvum oocysts in chlorinated tap water. Archives of Environmental Health, 59(9): 462–466

    Article  Google Scholar 

  • Liang B, Kong D, Ma J, Wen C, Yuan T, Lee D J, Zhou J, Wang A (2016). Low temperature acclimation with electrical stimulation enhance the biocathode functioning stability for antibiotics detoxification. Water Research, 100: 157–168

    Article  CAS  Google Scholar 

  • Liu Y, Dong S, Kuhlenschmidt M S, Kuhlenschmidt T B, Drnevich J, Nguyen T H (2015). Inactivation mechanisms of Cryptosporidium parvum oocysts by solar ultraviolet irradiation. Environmental Science. Water Research & Technology, 1(2): 188–198

    Article  CAS  Google Scholar 

  • Medeiros R C, Sammarro Silva K J, Daniel L A (2020). Wastewater treatment performance in microbiological removal and oocysts viability assessed comparatively to fluorescence decay. Environmental Technology, 1811396: 1–9

    Google Scholar 

  • Montemayor M, Valero F, Jofre J, Lucena F (2005). Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain. Journal of Applied Microbiology, 99(6): 1455–1462

    Article  CAS  Google Scholar 

  • Morita S, Namikoshi A, Hirata T, Oguma K, Katayama H, Ohgaki S, Motoyama N, Fujiwara M (2002). Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts. Applied and Environmental Microbiology, 68(11): 5387–5393

    Article  CAS  Google Scholar 

  • Nasser A M (2016). Removal of Cryptosporidium by wastewater treatment processes: A review. Journal of Water and Health, 14(1): 1–13

    Article  Google Scholar 

  • Nedwell D B (1999). Effect of low temperature on microbial growth: Lowered affinity for substrates limits growth at low temperature. FEMS Microbiology Ecology, 30(2): 101–111

    Article  CAS  Google Scholar 

  • Neumayerová H, Koudela B (2008). Effects of low and high temperatures on infectivity of Cryptosporidium muris oocysts suspended in water. Veterinary Parasitology, 153(3–4): 197–202

    Article  Google Scholar 

  • O’Hara S P, Chen X M (2011). The cell biology of cryptosporidium infection. Microbes and Infection, 13(8–9): 721–730

    Article  Google Scholar 

  • European Union (2020). Regulation (EU) 2020/741 of the European Parliament and of the Council of 25 May 2020 on minimum requirements for water reuse. Official Journal of the European Union. Brussels: European Union

    Google Scholar 

  • Okhuysen P C, Chappell C L (2002). Cryptosporidium virulence determinants—Are we there yet? International Journal for Parasitology, 32(5): 517–525

    Article  CAS  Google Scholar 

  • Phadtare S, Inouye M (2004). Genome-wide transcriptional analysis of the cold shock response in wild-type and cold-sensitive, quadruple csp-deletion strains of Escherichia coli. Journal of Bacteriology, 186(20): 7007–7014

    Article  CAS  Google Scholar 

  • Prasad A S B, Shruptha P, Prabhu V, Srujan C, Nayak U Y, Anuradha C K R, Ramachandra L, Keerthana P, Joshi M B, Murali T S, Satyamoorthy K (2020). Pseudomonas aeruginosa virulence proteins pseudolysin and protease IV impede cutaneous wound healing. Laboratory Investigation, 100(12): 1532–1550

    Article  CAS  Google Scholar 

  • Rajapandi T (2020). Apicomplexan lineage-specific polytopic membrane proteins in Cryptosporidium parvum. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, 44(2): 467–471

    Article  Google Scholar 

  • Ramamurthy T, Ghosh A, Pazhani G P, Shinoda S (2014). Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Frontiers in Public Health, 2: 103–111

    Article  Google Scholar 

  • Ramo A, Del Cacho E, Sánchez-Acedo C, Quílez J (2017). Occurrence and genetic diversity of Cryptosporidium and Giardia in urban wastewater treatment plants in north-eastern Spain. Science of the Total Environment, 598: 628–638

    Article  CAS  Google Scholar 

  • Ran Z, Li S, Huang J, Yuan Y, Cui C, Williams C D (2010). Inactivation of Cryptosporidium by ozone and cell ultrastructures. Journal of Environmental Sciences-China, 22(12): 1954–1959

    Article  CAS  Google Scholar 

  • Razzolini M T P, Breternitz B S, Kuchkarian B, Bastos V K (2020). Cryptosporidium and Giardia in urban wastewater: A challenge to overcome. Environmental Pollution, 257: 113545

    Article  CAS  Google Scholar 

  • Reid S D, Green N M, Buss J K, Lei B, Musser J M (2001). Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription. Proceedings of the National Academy of Sciences of the United States of America, 98(13): 7552–7557

    Article  CAS  Google Scholar 

  • Rider S D Jr, Zhu G (2010). Cryptosporidium: genomic and biochemical features. Experimental Parasitology, 124(1): 2–9

    Article  CAS  Google Scholar 

  • Rizk N, Herrawy A, Gad M, Shaheen M, Elmahdy E (2019). Existence and removal of Rotaviruses group A and Cryptosporidium species in a wastewater treatment plant. Polish Journal of Environmental Studies, 28(6): 4331–4339

    Article  CAS  Google Scholar 

  • Robertson L J, Paton C A, Campbell A T, Smith P G, Jackson M H, Gilmour M A, Black S E, Stevenson D A, Smith H V (2000). Giardia cysts and Cryptosporidium oocysts at sewage treatment works in Scotland, UK. Water Research, 34(8): 2310–2322

    Article  CAS  Google Scholar 

  • Ryan U, Power M (2012). Cryptosporidium species in Australian wildlife and domestic animals. Parasitology, 139(13): 1673–1688

    Article  Google Scholar 

  • Sammarro Silva K J, Sabogal-Paz L P (2021). Cryptosporidium spp. and Giardia spp. (oo)cysts as target-organisms in sanitation and environmental monitoring: A review in microscopy-based viability assays. Water Research, 189: 116590

    Article  CAS  Google Scholar 

  • Santos P R, Daniel L A (2017). Occurrence and removal of Giardia spp. cysts and Cryptosporidium spp. (oo)cysts from a municipal waste-water treatment plant in Brazil. Environmental Technology, 38(10): 1245–1254

    Article  CAS  Google Scholar 

  • Schmitz B W, Moriyama H, Haramoto E, Kitajima M, Sherchan S, Gerba C P, Pepper I L (2018). Reduction of Cryptosporidium, Giardia, and fecal indicators by bardenpho wastewater treatment. Environmental Science & Technology, 52(12): 7015–7023

    Article  CAS  Google Scholar 

  • Song J, Zhao J, Gao H, Liu Y, Yue H, Zhang J, Su Y, Yin H (2011). Serological detection of Cryptosporidium spp. infection in outpatients in Changchun. Chinese Journal of Parasitology and Parasitic Diseases, 29(3): 239–241 (in Chinese)

    Google Scholar 

  • Song X, Jing L (2015). Annual observations of climatic impacts in the Songhua River Basin, China. Advances in Meteorology, 2015: 1–12

    Article  CAS  Google Scholar 

  • Su Y (2011). Molecular epidemiology of Cryptosporidium and Giardia infection in a part of children and cattle in northeast China. Dissertation for the Master Degree. Changchun: Jilin University (in Chinese)

    Google Scholar 

  • Sulaiman I M, Morgan U M, Thompson R C, Lal A A, Xiao L (2000). Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene. Applied and Environmental Microbiology, 66(6): 2385–2391

    Article  CAS  Google Scholar 

  • Swaffer B A, Vial H M, King B J, Daly R, Frizenschaf J, Monis P T (2014). Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment. Water Research, 67: 310–320

    Article  CAS  Google Scholar 

  • Tai L, Li J, Yin J, Zhang N, Yang J, Li H, Yang Z, Gong P, Zhang X (2019). A novel detection method of Cryptosporidium parvum infection in cattle based on Cryptosporidium parvum virus 1. Acta Biochimica et Biophysica Sinica, 51(1): 104–111

    Article  CAS  Google Scholar 

  • Trevors J T, Bej A K, Mojib N, van Elsas J D, Van Overbeek L (2012). Bacterial gene expression at low temperatures. Extremophiles, 16(2): 167–176

    Article  CAS  Google Scholar 

  • Wanyiri J, Ward H (2006). Molecular basis of Cryptosporidium-host cell interactions: Recent advances and future prospects. Future Microbiology, 1(2): 201–208

    Article  CAS  Google Scholar 

  • Xiao F, Huang J H, Zhang B, Cui C (2009). Effects of low temperature on coagulation kinetics and floc surface morphology using alum. Desalination, 237(1–3): 201–213

    Article  CAS  Google Scholar 

  • Xiao L, Alderisio K, Limor J, Royer M, Lal A A (2000). Identification of species and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool. Applied and Environmental Microbiology, 66(12): 5492–5498

    Article  CAS  Google Scholar 

  • Xing B S, Guo Q, Jiang X Y, Chen Q Q, Li P, Ni W M, Jin R C (2016). Influence of preservation temperature on the characteristics of anaerobic ammonium oxidation (anammox) granular sludge. Applied Microbiology and Biotechnology, 100(10): 4637–4649

    Article  CAS  Google Scholar 

  • Xu S, Wu X, Lu H (2021). Overlooked nitrogen-cycling microorganisms in biological wastewater treatment. Frontiers of Environmental Science & Engineering, 15(6): 133

    Article  CAS  Google Scholar 

  • Zacharia A, Outwater A H, Ngasala B, Van Deun R (2018). Pathogenic parasites in raw and treated wastewater in Africa: A review. Resources and Environment, 8: 232–240

    Google Scholar 

  • Zahedi A, Gofton A W, Greay T, Monis P, Oskam C, Ball A, Bath A, Watkinson A, Robertson I, Ryan U (2018). Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing. Science of the Total Environment, 644: 635–648

    Article  CAS  Google Scholar 

  • Zhang T, Gao X, Wang D, Zhao J, Zhang N, Li Q, Zhu G, Yin J (2021). A single-pass type I membrane protein from the Apicomplexan parasite Cryptosporidium parvum with nanomolar binding affinity to host cell surface. Microorganisms, 9(5): 1015–1028

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Natural Science Foundation of China (Nos. 51908062 and 51978135). It was also supported by the Scientific and Technological Development Plan Project of Jilin Province (China) (No. 20200201042JC). We thank Blessing Ifeoluwa Ogunniran for her linguistic and editing assistance during the revision of this manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Wei Fan.

Additional information

Highlights

Cryptosporidium in WWTPs in a cold region was investigated in different seasons.

• The overall removal efficiency of Cryptosporidium in WWTPs was over 84%.

• The infectivity rate declined below 53% in effluents mainly due to disinfection.

• The infectivity of Cryptosporidium increased with a seasonal drop in temperature.

• Low temperature promotes binding protein retention and virulence genes expression.

Special Issue—China Urban Water Environment and Water Ecology

Supporting Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xiao, D., Lyu, Z., Chen, S. et al. Tracking Cryptosporidium in urban wastewater treatment plants in a cold region: Occurrence, species and infectivity. Front. Environ. Sci. Eng. 16, 112 (2022). https://doi.org/10.1007/s11783-022-1533-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11783-022-1533-8

Keywords

Navigation