Occurrence of Cryptosporidium and Giardia in recycled waters used for irrigation and first description of Cryptosporidium parvum and C. muris in Greece

Abstract

Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

Performance of three small-scale wastewater treatment plants. A challenge for possible re use

“…Carducci and Verani showed the presence of adenovirus DNA in 100 % of collected samples, while an average reduction of 2 log was recorded (Carducci and Verani 2013). Similarly, AdVs were detected in 100 % of wastewater and sewer overflows (CSOs) For Cryptosporidium and Giardia spp., please refer to Spanakos et al (2015); units: E. coli, log 10 MPN/L; TC, log 10 MPN/L; bacteriophages, log 10 plaque forming units (PFU)/L; hAdVs, log 10 GC/L discharge samples in the study of Fong et al (2010). Average adenovirus DNA concentrations in sewage and CSOs were 1.15×10(6) viruses/liter and 5.35×10(5) viruses/liter, respectively, while the adenovirus removal was calculated at <2 log(10) (99 %) for this WWTP (Fong et al 2010).…”

“…One of the sources of these parasites can be treated wastewater from wastewater treatment plants (Hachich et al 2013;Sroka et al 2013). In an attempt to enrich poor existing data on the prevalence of these parasites in wastewater effluents in Greece, the study produced very interesting data from the parasitological analysis of the sewage samples of the study (Spanakos et al 2015).…”

“…Sewage samples were analyzed for total coliforms and E. coli, using the multiple tube method (CEN BT/TF 151 -PrEN 15214-3), bacteriophages, using the double-layer method (ISO 10705-2:1000), Salmonella spp., using an ISO method (DRAFT ISO 19250:2009 (E)), molds, Candida, and Pseudallescheria boydii, according to previously published methods (Katragkou et al 2007;Cortez et al 2008;Stripeli et al 2009), helminths according to APHA standard method (2012), and Cryptosporidium and Giardia, as described by Spanakos et al (2015).…”

Request for scientific and technical assistance on proposed EU minimum quality requirements for water reuse in agricultural irrigation and aquifer recharge

“…In the JRC draft report it is indicated that information is often not available on concentrations, dose-response relationships and disease burden for various waterborne pathogens in treated wastewater in Europe. However, the EFSA experts have identified various scientific papers addressing some of the most relevant pathogens (Montemayor et al, 2005;Levantesi et al, 2010;Rodríguez-Manzano et al, 2012;Rusiñol et al, 2015;Spanakos et al, 2015;Eftim et al, 2017). This should by no means be considered as an extensive literature search on the topic, but is given as an illustration that relevant data are available that address the previous point.…”

“…Robertson et al (2006). Furthermore, studies from Europe (Greece and Spain) also describe the common occurrence of Cryptosporidium in tertiary-treated wastewater (Montemayor et al, 2005;Spanakos et al, 2015).…”