Giardia cysts in wastewater treatment plants in Italy

Abstract

Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O(2) and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P = 0.05, analysis of variance). To characterize the cysts at the molecular level, the beta-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.

FIG. 1.

Number of Giardia cysts per liter in wastewater samples collected at different steps in the treatment process at treatment plants in Bergamo (plant 1), Naples (plant 2), Cagliari (plant 3), and Palermo (plant 4) in the spring, summer, autumn, and winter of the year 2000. Solid bars, influent samples; thick striped bars, samples after primary treatment; gray bars, samples after secondary treatment; white bars, effluent samples. In plant 1, the thin striped bars show the number of Giardia cysts shortly after oxidation with O₂ had begun, and the white bars show the number of Giardia cysts after oxidation with O₂ and sedimentation was completed. In plant 4, no samples were collected after primary treatment.

FIG. 2.

Electrophoretic separation of β-giardin amplification products from wastewater samples. Lanes 1 to 3, influent samples from the plant 1; lanes 4 to 6, influent samples from plant 2; lane M, 100-bp molecular ladder; lanes 7 to 9, influent samples from plant 3; lanes 10 and 11, influent samples from plant 4; lane 12, negative control; lane 13, positive control.

FIG. 3.

Electrophoretic separation of β-giardin PCR products after restriction with the endonuclease HaeIII. Lanes M, 50-bp molecular ladder; lane 1, positive control for assemblage A; lane 2, positive control for assemblage B; lanes 3 to 6, samples of influent from plant 1; lanes 7 to 10, samples of influent from plant 2; lanes 11 to 14, samples of influent from plant 3; lane 15 to 18, samples of influent from plant 4. For each plant, influent samples from spring, summer, autumn, and winter were typed and are shown in that order. Note the concomitant presence of restriction fragments specific for assemblages A and B in samples 3, 4, 5, 10, 11, 13, 16, and 18.