Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a significant public health concern. The aim of the present study was to demonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the present study with SAB results reflecting SW and BW results. Further highlights were demonstrated in the potential of using low-volume samples for the detection of parasites in source water. Scanning electron microscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).

Cryptosporidium parvum oocysts may reach soil through direct deposition of human or animal fecal material, irrigation with raw wastewater or untreated effluents, and contaminated runoff. Examination of soil samples for oocyst presence is of primary importance in order to prevent secondary contamination of crops and groundwater. Several methods were proposed for oocyst recovery from soil samples; however, their efficiency was very low. In the present study, four known methods used to recover oocysts from water and fecal samples (sedimentation, sedimentation with reduced water content, sucrose floatation, water-ether separation) were compared to a method used in the past to recover bacterial spores from bottom sediments (two-phase separation). The two-phase separation technique proved to be the best method of choice resulting in a recovery average of 61.2 ± 15.6%. According to this method, the lowest and highest recoveries were 37% to 95%, respectively. Two other important outcomes were observed with the soil experimental set-up: (1) recovery efficiency is influenced by oocyst viability (high viability was directly correlated with increased recovery efficiency) and (2) high sand content of soil samples reduced oocyst recovery by its detrimental effect on oocyst viability.

Treatments using ozone for filter backwash water (FBW) and calcium oxide for floated residue (FR) were evaluated adopting bench-scale testing for the inactivation of Giardia spp. cysts and Cryptosporidium parvum oocysts. The protocol chosen for protozoa detection involved following the concentration step by direct centrifugation (adding ICN 7X cleaning solution at 1.0%) and purification by immunomagnetic separation (IMS). The FR treatment with calcium oxide (dosage of 23 mg CaO 100 mL⁻¹ and 3-day contact time at 25 °C) proved to be efficient, as no parasites were detected after the treatment. The reduction of calcium oxide dosage (16 mg CaO 100 mL⁻¹ and 3-day contact time at 25 °C) was insufficient to inactivate the protozoa, since potentially viable organisms were identified using propidium iodide (PI). Concerning the disinfection conditions with ozone (5-min and 10-min contact time and dosage of 10 mg O₃ L⁻¹ and 7.5 mg O₃ L⁻¹, respectively), there was complete removal of the target organisms, as no protozoa were detected after the FBW treatment. From the results obtained, the tested treatments can be considered promising alternatives for water treatment plants (WTPs). However, the costs incurred from these treatments have to be considered.

The present study aimed to look at the fate of protozoan parasite Cryptosporidium parvum oocysts applied through surface drip irrigation on reclaimed water irrigation-history and non-history sandy-loam (Hamra) soil columns. A new and simple isolation method for recovery of oocysts from soil samples was developed and used along this study. The new soil isolation method of oocysts is based on the “two phase separation method” formerly used to recover Clostridium perfringens spores from sediments and soil samples with minor modifications. The range recovery achieved by this method was 64-95% (mean 61.2 ± 17.4). The objectives of the second part of this study were to investigate several physical and chemical factors governing transport and survival of C. parvum oocysts in sandy-loam soil columns by breakthrough curves. Comparison of fresh water and reclaimed water irrigation revealed that reclaimed water irrigated-history soil was more hydrophobic allowing water flow through channels with poor oocysts retention and fast flow. Examination of the organic matter effect (originating from reclaimed water irrigation) on oocysts breakthrough revealed that their soil infiltration increased. Calculations of oocysts concentration at different columns depths showed that most of the oocysts were retained in the first 5 cm of soil column. In the present study, comparing the two soil types (history and non-history of effluents irrigation) beside the surface electrostatic charge, one of the main elements found to affect oocysts infiltration and transport in soil columns was soil hydrophobicity caused by soluble organic matter originating from reclaimed water irrigation. Therefore, prior to application in soil irrigation, reclaimed water should be treated to high quality (i.e. membrane technology as the best option) to prevent enhanced transport of various pathogens through those irrigated soils.

Toxoplasma gondii, Cryptosporidium parvum, and Giardia duodenalis are human waterborne protozoa. These worldwide parasites had been detected in various watercourses as recreational, surface, drinking, river, and seawater. As of today, water protozoa detection was based on large water filtration and on sample concentration. Another tool like aquatic invertebrate parasitism could be used for sanitary and environmental biomonitoring. In fact, organisms like filter feeders could already filtrate and concentrate protozoa directly in their tissues in proportion to ambient concentration. So molluscan shellfish can be used as a bioindicator of protozoa contamination level in a site since they were sedentary. Nevertheless, only a few researches had focused on nonspecific parasitism like protozoa infection on aquatic invertebrates. Objectives of this review are twofold: Firstly, an overview of protozoa in worldwide water was presented. Secondly, current knowledge of protozoa parasitism on aquatic invertebrates was detailed and the lack of data of their biological impact was pointed out.