Ocurrence Of Three Common Waterborne Pathogens In Raw Water,Wastewater And Overflow In Shanghai

Cryptosporidium spp.,Giardia duodenalis,and Enterocytozoon bieneusi are three kinds of important waterborne pathogens and caused more than 90%waterborne outbreak incidents.However,investigations aiming the role of raw water,wastewater and overflow in the transmission of these pathogens in developing country is scarce.The present study used molecular tools to genotyped the Cryptosporidium spp.,G.duodenalis and E.bieneusi and analyzed the characteristics of contamination in raw water,wastewater,and overflow in Shanghai.In addition,the present study estimated the pathogenic risk of these parasites.The raw water and wastewater samples were preprocessed by flocculation or centrifugation,respectively.To obtain the positive rate and the species or genotypes of the Cryptosporidium spp.,G.duodenalis and E.bieneusi,Nested-PCR combined sequencing was conducted at SSU rRNA,tpi,bg,gdh and ITS gene,respectively.The positive samples for Cryptosporidium spp.were furtherly analyzed to subtype in gp60 locus.To determine the subtypes of('ryptosporidium in multilocus,positive samples were analyzed by multilocus subtyping method in different loci.To investigate the distribution of these waterborne parasites in raw water in Shanghai,106 samples were collected during the dead pig floating incident outbroke in 2013,72 samples were collected after that incident and 78 samples were collected in 2016 when the Jinze reservoir was built.Of these samples,we detected the G.duodenalis in all samples and characterized Cryptosporidium spp.and E.bieneusi in the samples collected in 2016.The earlier research indicated that the positive rate of Cryptosporidium spp in 2013 and 2014 were 53.8%and 13.9%,respectively.E.bieneusi were 33.9%and 27.8%,respectively.The present study indicated that the positive rate of G.duodenalis in raw water of Huangpu river in 2013,2014 and 2016 were 22.6%,13.9%,and 5.1%,respectively.Meanwhile,the positive rate of Cryptosporidium spp.and E.bieneusi were 0%and 1.3%.The results indicated that the positive rates of these pathogens were declined yearly and the floating incident of dead pigs has caused severe contamination.In addition,the built of Jinze reservoir was one of the possible reason that reduced the positive rate of these pathogens in the Huangpu river.Moreover,the major genotypes of G.duodenalis and E.bieneusi in raw water were A2 and D,these genotypes were zoonotic.As a consequence,these parasites in raw water were latent threat to local citizens.To unravel the distribution and pathogenic potential of these waterborne pathogens,110 wastewater samples were collected from two wastewater treatment plants(WWTPs)and two wastewater pump stations(WWPSs).The positive rate of Cryptosporidiun spp.,G.duodenalis and E.bieneusi were 7.3%,82.5%,and 98.2%,respectively.Totally,three Cryptosporidium species,three G.duodenalis genotypes,and nine E.bieneusi genotypes were characterized.Of these species or genotypes,Cryptosporidium hominis,G.duodenalis genotype A2,A3 and assemblage B,E.bieneusi genotype D could infect humans.These results indicated that the wastewater could contaminate the water source if handled improperly.Since the wastewater would be charged into the streamway as overflow instead of treatment plant system when encountered heavy rain,the present study investigated the positive rate and genotypes of these pathogens in combined sewer overflows(CSOs)in Shanghai.Moreover,we compared the overflow and wastewater simultaneously.Totally,40 samples were collected from two WWPSs encounted the heavy rains and 40 samples were collected from two WWPSs after the rain.The positive rates of Cryptosporidium in overflow and wastewater were 30%and 37.5%,G.duodenalis were 82.5%,and 80.0%,E.bieneusi were 92.5%and 100.0%.Totally,10 Cryptosporidium species,three G.duodenalis assemblages,and eight E.bieneusi genotypes were characterized.Besides Cryptosporidium hominis and Cryptosporidium parvum,two new emerging species were identified:Cryptosporidium ubiquitum and Cryptosporidium viatorum.Two novel C.ubiquitum subtype families were identified.The major genotype of E.bieneusi and G.duodenalis in overflow and wastewater were group 1 and subassemblage A?.These genotype and subassemblage are known human pathogens.Compared to the results obtained from wastewater,high positive rate and zoonotic characteristic of overflow indicated that the overflow was the latent source of these pathogens in surface waters.The aforementioned results confirmed the existence of zoonotic waterborne pathogens in raw waters,wastewater and combined sewer overflows in Shanghai.The overflow was severely contaminated by these pathogens and posed pathogenic risks.The present study has provided the theoretical foundation for understanding the environmental transmission of these parasites in kinds of waters and the effect on the quality of drinking water in Shanghai.