In order to establish seawater contamination by emerging protozoan parasites, we used qPCR to molecularly characterize and evaluate the parasitic burden of Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii, and Cyclospora cayetanensis in 1255 wild bivalve mollusks collected along the Tunisian coasts. T. gondii, G. duodenalis and C. cayetanensis were detected in 6.9% (99% CI=1.6–12.2%) pools of Ruditapes decussatus. None of the samples were found positive to Cryptosporidium spp.; 6.6% pools of R. decussatus were positive for T. gondii Type I, 1.6% for G. duodenalis assemblage A, and 1.6% for the association T. gondii Type I/C. cayetanensis/G. duodenalis assemblage A. R. decussatus harbored up to 77500 oocysts/sample of T. gondii, up to 395 cysts/sample of G. duodenalis, and 526 oocysts/sample of C. cayetanensis. These results provide the first evidence that the Tunisian coasts are contaminated by zoonotic protozoan parasites that can constitute a direct or indirect risk for human health.

The effects of precipitation on the hygienic quality of water and blue mussels collected from five different localities in the urban areas in the Inner Oslofjord were investigated, with samples analysed for Escherichia coli, Salmonella spp., pathogenic Vibrio spp., Norovirus, Sapovirus, Cryptosporidium spp. and Giardia duodenalis. The sampling sites were located at varying distances from the outlet of combined sewer overflows (CSO)-impacted rivers/streams. In general, 1–3 log10 increases in fecal indicator bacteria and human pathogens were observed after heavy rainfalls. Blue mussels appeared to be a useful indicator of the impact of sewage at these sites, and generally a good correlation was identified between concentrations of E. coli and other human pathogens in the mussels. Provision of general advice to the public of avoiding areas near the outlets of CSO-impacted rivers after heavy rainfall may reduce the risk of gastroenteritis by bathers and others that may swallow water during recreational activities.

The main objective of this study was to characterize the Giardia duodenalis isolates from Iranian patients in Fars Province, south of Iran by biochemical and molecular methods. Fifteen mass cultivated of G. duodenalis isolates in modified TYI-S-33 medium were analyzed using isoenzyme electrophoresis and PCR genotyping. Polyacrylamide gel electrophoresis (PAGE) of five different enzyme systems was used to characterize isolates: (i) glucose-6-phosphate dehydrogenase, (ii) glucose phosphate isomerase, (iii) malate dehydrogenase, (iv) malic enzyme, and (v) phosphoglucomutase. As well, a fragment of the SSU-rDNA (292 bp) gene was amplified by PCR using the primers RH11 and RH4. The sequencing of the PCR products and phylogenetic tree were performed. The isoenzyme electrophoretic profiles divided fifteen G. duodenalis isolates into four zymodemes. G6PD, GPI, MDH, ME, and PGM enzyme systems showed 1, 2, 2, 3, and 3 enzyme pattern, respectively. G6PD isoenzyme pattern had the most homogeneity, while isoenzyme patterns of ME and PGM had the most heterogeneity in our study. Genotyping results indicated that the zymodemes 1–4 were categorized in assemblage A based on the SSU-rDNA gene. Phylogenetic analysis showed that all four zymodemes were distributed within the cluster of assemblage A. Our results indicated that both isoenzyme and DNA analyses were useful to characterize the isolates of Giardia and distinguishing various zymodemes and assemblages. It could be suggested that the genetic diversity among isoenzymes profiles of G. duodenalis may explain the variable clinical manifestations, pathogenicity, host response, drug susceptibility, and treatment efficacy of human giardiasis.

Cryptosporidium and Giardia are associated with cases of water and foodborne outbreaks in the world. This study included 50 samples of surface raw water collected from two watersheds in the state of São Paulo, Brazil. The isolation of (oo)cysts was performed in accordance with the U.S. Environmental Protection Agency’s methods 1623 and genotypic characterization and quantification were carried out by Nested PCR and qPCR assays based on 18S rRNA and gdh genes, respectively. U.S. EPA 1623 method showed the presence of (oo)cysts in 40% ([Formula: see text] = 0.10 oocysts/L) and 100% ([Formula: see text] = 7.6 cysts/L) of samples from São Lourenço River, respectively, and 24% ([Formula: see text] = 0.8 oocysts/L) and 60% ([Formula: see text] = 1.64 cysts/L) of Guarapiranga Reservoir, respectively. The qPCR assay detected C. hominis/parvum in 52% (0.06 to 1.85 oocysts/L) of São Lourenço River and 64% (0.09 to 1.4 oocysts/L) of Guarapiranga Reservoir samples. Presence/absence test for Giardia intestinalis was positive in 92% of São Lourenço River and 8% of Guarapiranga Reservoir samples. The assemblage A was detected in 16% (0.58 to 2.67 cysts/L) in São Lourenço River and no positive samples were obtained for assemblage B in both water bodies. The characterization of anthroponotic species C. parvum/hominis, G. intestinalis, and assemblage A was valuable in the investigation of possible sources of contamination in the watersheds studied confirming the need of expanding environmental monitoring measures for protection of these water sources in our country.

Giardia and Cryptosporidium are potentially pathogenic protozoa which are ubiquitous in ambient surface water. The present study included 60 samples of surface water from three sampling sites from the Rímac River, Lima and Callao, Peru, to detect the occurrence of Giardia spp. and Cryptosporidium spp. and to perform molecular characterization of specimens found. Water samples were concentrated using the membrane filtration technique, and following elution, cysts and oocysts were visualized by direct immunofluorescence assay (IFA). For molecular characterization, tpi and bg gene fragments and 18S rRNA were amplified by nested PCR for Giardia and Cryptosporidium, respectively, followed by sequencing and phylogenetic analysis. Giardia cysts were found in 93.3% of the analyzed samples, whereas Cryptosporidium oocysts were detected in 15%. The positivity of the Giardia cysts was 86.6% (n = 26) in 2014, while Cryptosporidium oocysts were not detected. In 2015, both protozoa were found in raw water samples, with all 30 samples collected positive for Giardia cysts (100.0%) and 9 positive for Cryptosporidium oocysts (30.0%). Oocysts were detected in 20.0% of water samples from sites 1 (mean 5.25 oocysts/L) and 2 (mean 52.3 oocysts/L), while at site 3, oocysts were detected in 50.0% of raw water samples (mean 193.6 oocysts/L). The presence of Giardia duodenalis assemblage A was confirmed in several samples by the phylogenetic positioning of the bg and tpi genes, and the sub-assemblage AII was predominant (8/9). Sequencing for Cryptosporidium resulted in profiles compatible with Cryptosporidium hominis, Cryptosporidium meleagridis, and Cryptosporidium baileyi. This is the first time that the presence of G. duodenalis assemblage A/sub-assemblage AII and Cryptosporidium species has been reported in surface water samples in Peru. These Cryptosporidium species and the Giardia duodenalis assemblage are associated with human disease which highlights the potential risk to public health and the need to increase environmental monitoring measures to protect this water body.

A total of 54 raw wastewater samples collected from three urban treatment plants and two slaughterhouses in Tehran, Iran, were assessed for the presence of the Giardia cysts using immunofluorescence with monoclonal antibodies. To characterize the cysts at the molecular level, the three genetic loci were amplified and sequenced. The assemblages A (37.5 %) and E (58.3 %) were detected in livestock wastewater samples. Assemblage A, which is composed of only G. duodenalis genotype, was detected in 100 % of urban wastewater samples. The subassemblages A2, A3, A-I, A-II, and E3 were identified with β-giardin, triose phosphate isomerase, and glutamate dehydrogenase genes. This study is the first to report on G. duodenalis genotypes in aquatic environmental samples in Iran.

The objectives of the study were to detect and genotype Cryptosporidium spp. and Giardia intestinalis in wastewater samples obtained from five cities with high transit of people in the State of São Paulo, Brazil, and at the entrance of a Wastewater Treatment Plant (WWTP) in Lima, Peru. Samples were collected and concentrated by centrifugation. The genomic DNA was extracted for molecular characterization by nested PCR for Cryptosporidium and double nested PCR for Giardia, followed by sequencing and phylogenetic analysis. G. intestinalis was found in 63.6 % of the samples, and the human assemblages A and B were identified. Cryptosporidium sp. was found in 36.4 % of the samples, and the species were corresponding to Cryptosporidium hominis, Cryptosporidium cuniculus, and Cryptosporidium muris. Results revealed the presence of human pathogenic Cryptosporidium species and G. intestinalis human pathogenic assemblages. Molecular tools highlight the importance to map the genetic diversity of these parasites, as well as to detect their epidemiological circulation pathway in the environment.

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.