Occurrence of Cryptosporidium oocysts and Giardia cysts in effluent from sewage treatment plant from eastern Poland
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Department of Parasitology, National Veterinary Research Institute, Puławy, Poland
Department of Zoonoses, Institute of Rural Health, Lublin, Poland
Jacek Sroka   

Department of Parasitology, National Veterinary Research Institute, Puławy, Poland
Ann Agric Environ Med. 2013;20(Special Issue 1):57–62
Cryptosporidium spp] and Giardia lamblia (synonyms: Giardia duodenalis, Giardia intestinalis) are emerging protozoa causing disease in humans and animals worldwide. These parasites can pose a serious threat to immunocompromised people, for whom the symptoms are more severe and may include abdominal pain, watery diarrhoea, nausea, headaches, malaise, and fever. One of the sources of these parasites can be treated wastewater from wastewater treatment plants (WTPs). Samples of treated wastewater (effluent), each of 10 L volume, were collected from 13 municipal WTPs located in eastern Poland. Cryptosporidium oocysts and Giardia cysts were separated by the immunomagnetic method. The presence and/or concentration of protozoan (oo)cysts in effluent samples were determined by direct immunofluorescent microscopy, nested PCR and Real Time PCR. Viability of (oo)cysts was determined by double-staining with the use of Live/Dead BacLight kit (Invitrogen). Cryptosporidium spp. oocysts were detected in 8 WTPs (61.5%) and Giardia spp. cysts in 11 WTPs (84.6%) by microscopic analysis. Both pathogens were detected in samples from 7 WTPs. Median concentrations of Cryptosporidium and Giardia (oo)cysts in 13 examined samples were 2.2/L and 6.6/L, respectively, while mean concentrations were 28.5/L and 113.6/L, respectively. In positive samples, Cryptosporidium oocysts concentrations ranged from 0.4 – 154.1 oocysts per litre, and Giardia cysts concentrations ranged from 0.7 – 660 cysts per litre. By nested PCR, Giardia DNA was detected in 4 samples of the 13 examined, (30.8%) while Cryptosporidium DNA was never detected. In Real Time PCR, positive results for Giardia were obtained in 5 samples (38.5%) and in none of the samples for Cryptosporidium, with the exception of one equivocal result. Viable (oo)cysts of Cryptosporidium and Giardia were detected in 3 out of 4 samples examined, in the ranges of 12.5 – 60% and 50 – 100% of total (oo)cysts, respectively. In view of our preliminary study, the presence of oocysts and cysts (largely viable) in effluents from WTPs imply a risk of transmission of waterborne protozoan parasites to humans. Therefore, additional wastewater purification procedures are necessary.
Fayer R. Cryptosporidium: a water-borne zoonotic parasite. Vet Parasitol. 2004; 126: 37–56.
Reynolds KA, Mena KD, Gerba CP. Risk of waterborne illness via drinking water in the United States. Rev Environ Contam Toxicol. 2008; 192: 117–158.
Smith HV, Caccio SM, Cook N, Nichols RAB, Tait A. Cryptosporidium and Giardia as foodborne zoonoses. Vet. Parasitol. 2007; 149: 29–40.
Guy RA, Payment P, Krull UJ, Horgen PA. Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl Environ Microbiol. 2003; 69: 5178–5185.
Ryan U, Xiao L, Read C, Zhou L, Lal AA, Pavlasek I. Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl Environ Microbiol. 2003; 69: 4302–4307.
Thompson RCA. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int J Parasitol. 2000; 30: 1259–1267.
Thompson RCA, Armson A, Ryan UM. Cryptosporidium: from molecules to disease. Elsevier, Amsterdam 2003.
Karanis P, Kourenti C. Waterborne transmission of protozoan parasites: a review of world-wide outbreaks. In: Fourth International Giardia Conference and First Combined Giardia-Cryptosporidium Meeting, 20–24 September 2004, Amsterdam, The Netherlands.
Karanis P, Papadopoulou C, Kimua A, Economou E, Kourenti C, Sakkas H. Cryptosporidium and Giardia in natural, drinking and recreational water of northwestern Greece. Acta Hydrochim Hydrobiol. 2002; 30: 49–58.
Mayer CL, Palmer CJ. Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater. Appl Environ Microbiol. 1996; 62: 2081–2085.
Nygård K, Schimmer B, Søbstad Ø, Walde A, Tveit I, Langeland N, Hausken T, Aavitsland P. A large community outbreak of waterborne giardiasis-delayed detection in a non-endemic urban area. BMC Public Health. 2006; 6:141.
Thompson RCA, Monis PT. Variation in Giardia: implications for taxonomy and epidemiology. Adv Parasitol. 2004; 58: 69–137.
Carrington EG, Gray P. The influence of cattle waste and sewage effluent on the levels of Cryptosporidium oocysts in surface waters. Foundation for Water Research, Marlow, Bucks, UK, 1993: 205–208.
Caccio SM, De Giacomo M, Aulicino FA, Pozio E. Giardia cysts in wastewater treatment plants in Italy. Appl Environ Microbiol. 2003; 69: 3393–3398.
Quintero-Betancourt W, Gennaccaro AL, Scott TM, Rose JB. Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents. Appl Environ Microbiol. 2003; 69: 5380–5388.
Reinoso R, Blanco S, Torres-Villamizar LA, Bécares E. Mechanisms for parasites removal in a waste stabilisation pond. Microb Ecol. 2011; 61: 684–692.
Cheng HW, Lucy FE, Graczyk TK, Broaders MA, Tamang L, Connolly M. Fate of Cryptosporidium parvum and Cryptosporidium hominisoocysts and Giardia duodenalis cysts during secondary wastewater treatments. Parasitol Res. 2009; 105:689–696.
Neto RC, Santos LU, Franco RM. Evaluation of activated sludge treatment and the efficiency of the disinfection of Giardia species cysts and Cryptosporidium oocysts by UV at a sludge treatment plant in Campinas, south-east Brazil. Water Sci Technol. 2006; 54: 89–94.
Wiandt S, Grimason AM, Baleux B, Bontoux J. Efficiency of wastewater treatment plants at removing Giardia sp. cysts in southern France. Schriftenr Ver Wasser Boden Lufthyg. 2000; 105: 35–42.
Rodriguez-Manzano J, Alonso JL, Ferrús MA, Moreno Y, Amorós I, Calgua B, Hundesa A, Guerrero-Latorre L, Carratala A, Rusiñol M, Girones R. Standard and new faecal indicators and pathogens in sewage treatment plants, microbiological parameters for improving the control of reclaimed water. Water Sci Technol. 2012; 66:2517–2523.
Chauret C, Springthorpe S, Sattar S. Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Can J Microbiol. 1999; 45: 257–262.
Gray NF. Biology of wastewater treatment. Imperial College Press, London, UK, 2004.
Lim YA, Wan Hafiz WI, Nissapatorn V. Reduction of Cryptosporidium and Giardia by sewage treatment processes. Trop Biomed. 2007; 24: 95–104.
Graczyk TK, Lucy FE, Tamang L, Miraflor A. Human enteropathogen load in activated sewage sludge and corresponding sewage sludge end products. Appl Environ Microbiol. 2007; 73: 2013–2015.
World Health Organization, Guidelines for Drinking Water Quality; Addendum-Microbial Agents in Drinking Water. 2nd Ed. Geneva 2002.
Lisle JT, Rose JB. Cryptosporidum contamination of water in the USA and UK: a minireview. J Water Supply Res Technol. 1995; 44: 103–105.
Robertson LJ, Paton CA, Campbell AT, Smith PG, Jackson MH, Gilmour RA, Black SE, Stevenson DA, Smith HV. Giardia cysts and Cryptosporidium oocysts at sewage treatment works in Scotland, UK. Water Res. 2000; 34: 2310–2322.
US EPA method 1623, 2001. Cryptosporidium and Giardia in water by filtration/IMS/FA. EPA-821-R-01–025, pp. 1–58.
Taghi-Kilani R, Gyürék LL, Millard PJ, Finch GR, Belosevic M. Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation. Int J Parasitol. 1996; 26: 637–646.
Caccio SM, De Giacomo M, Pozio E. Sequence analysis of the β-giardin gene and development of a polymerase chain reaction–restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 2002; 32: 1023–1030.
Xiao L, Lal AA, Jiang J. Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP. Methods Mol Biol. 2004; 268: 163–176.
Santın M, Trout JM, Xiao L, Zho L, Greiner E, Fayer R. Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet Parasitol. 2004; 122: 103–117.
Lonigro A, Pollice A, Spinelli R, Berrilli F, DiCave D, D’Orazi C, Cavallo P, Brandonisio O. Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation. Appl Environ Microbiol. 2006; 72: 7916–7918.
Pollice A, Lopez A, Laera G, Rubino G, Lonigro A. Tertiary filtered municipal wastewater as alternative water source in agriculture: a field investigation in southern Italy. Sci Total Environ. 2004; 324: 201–210.
Rozporządzenie Ministra Zdrowia z 29 marca 2007 r. w sprawie jakości wody przeznaczonej do spożycia przez ludzi (Dz. U. nr 61, poz. 417).
Ustawa z dnia 18 lipca 2001 r. Prawo wodne (Dz. U. z 2005 r. Nr 239, poz. 2019 z późn. zm.).
Payment P, Franco E. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts. Appl Environ Microbiol. 1993; 59: 2418–2424.
deRegnier D, Cole L, Schupp DG, Erlandsen SL. Viability of Giardia cysts suspended in lake, river and tap water. Appl Environ Microbiol. 1989; 55: 1223–1229.
Robertson LJ, Campbell AT, Smith HV. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl Environ Microbiol. 1992; 58: 3494–3500.
Whitmore TN, Robertson LJ. The effect of sewage sludge treatment processes on oocysts of Cryptosporidium parvum. J Appl Bacteriol. 1995; 78: 34–38.
Castro-Hermida JA, Garcıa-Presedo I, Almeida A, Gonzalez-Warleta M, Correia Da Costa JM, Mezo M. Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: A health risk for humans and animals. Water Research. 2009; 43: 4133–4142.
Dungeni M, Momba MNB. The abundance of Cryptosporidium and Giardia spp. in treated effluents produced by four wastewater treatment plants in the Gauteng Province of South Africa. Water SA 2010; 36: 425.
Tonani KA, Padula J, Julião FC, Fregonesi BM, Alves RI, Sampaio CF, Beda CF, Hachich E, Segura-Muñoz S. Persistence of Giardia, Cryptosporidium, Rotavirus and Adenovirus in Treated Sewage in São Paulo State, Brazil. J Parasitol. 2013 May 29. [Epub ahead of print].
Ben L, Khouja A, Cama V, Xiao L. Parasitic contamination in wastewater and sludge samples in Tunisia using three different detection techniques Parasitol Res. 2010; 107: 109–116.
Karanis P, Sotiriadou I, Kartashev V, Kourenti C, Tsvetkova N, Stojanova K. Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria. Environ. Res. 2006; 102: 260–271.
Plutzer J, Karanis P, Domokos K, Törökné A, Márialigeti K. Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes. Int J Hyg Environ Health. 2008; 211: 524–533.
Rimhanen-Finne R, Ronkainen P, Hänninen ML. Simultaneous detection of Cryptosporidium parvum and Giardia in sewage sludge by IC-PCR. J Appl Microbiol. 2001; 91: 1030–1035.
Gennaccaro AL, McLaughlin MR, Quintero-Betancourt W, Huffman DE, Rose JB. Infectious Cryptosporidium parvum oocysts in final reclaimed effluent. Appl Environ Microbiol. 2003; 69: 4983–4984.
McHarry MJ. Detection of Giardia in sewage effluent. J Protozool. 1984; 31: 362–364.
Lalancette C, Généreux M, Mailly J, Servais P, Côté C, Michaud A, Di Giovanni GD, Prévost M. Total and infectious Cryptosporidium oocyst and total Giardia cyst concentrations from distinct agricultural and urban contamination sources in Eastern Canada. J Water Health. 2012; 10:147–160.
McCuin RM, Clancy JL. Occurrence of Cryptosporidium oocysts in US wastewaters. J Water Health. 2006; 4: 437–452.
Bonadonna L, Briancesco R, Ottaviani M, Veschetti E. Occurrence of Cryptosporidium oocysts in sewage effluents and correlation with microbial, chemical and physical water variables. Environ Monit Assess. 2002; 75: 241–252.
Alonso JL, Amorós I, Cañigral I. Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples. Appl Microbiol Biotechnol. 2011; 89:1203–1211.
Robertson LJ, Hermansen L, Gjerde BK. Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway. Appl Environ Microbiol. 2006; 72:5297–5303.
Sturbaum GD, Reed C, Hoover PJ, Jost BH, Marshal MM, Sterling CR. Species-Specific, Nested PCR Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum oocysts. Appl Environ Microbiol. 2001; 67: 2665–2667.
Sulaiman IM., Jiang J, Singh A, Xiao L. Distribution of Giardia duodenalis genotypes and subgenotypes in raw urban wastewater in Milwaukee, Wisconsin. Appl Environ Microbiol. 2004; 70: 3776–3780.
Wallis PM, Erlandsen SL, Isaac-Renton JL, Olson ME, Robertson WJ, van Keulen H. Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada. Appl Environ Microbiol. 1996; 62:2789–2797.