Environmental Transport of Emerging Human-Pathogenic Cryptosporidium Species and Subtypes through Combined Sewer Overflow and Wastewater

Abstract

The environmental transport of <i>Cryptosporidium</i> spp. through combined sewer overflow (CSO) and the occurrence of several emerging human-pathogenic <i>Cryptosporidium</i> species in developing countries remain unclear. In this study, we collected 40 CSO samples and 40 raw wastewater samples from Shanghai, China, and examined them by PCR and DNA sequencing for <i>Cryptosporidium</i> species (targeting the small subunit rRNA gene) and <i>Giardia duodenalis</i> (targeting the triosephosphate isomerase, β-giardin, and glutamate dehydrogenase genes) and <i>Enterocytozoon bieneusi</i> (targeting the ribosomal internal transcribed spacer) genotypes. Human-pathogenic <i>Cryptosporidium</i> species were further subtyped by sequence analysis of the 60-kDa glycoprotein gene, with additional multilocus sequence typing on the emerging zoonotic pathogen <i>Cryptosporidium ubiquitum. Cryptosporidium</i> spp., <i>G. duodenalis</i>, and <i>E. bieneusi</i> were detected in 12 and 15, 33 and 32, and 37 and 40 CSO and wastewater samples, respectively, including 10 <i>Cryptosporidium</i> species, 3 <i>G. duodenalis</i> assemblages, and 8 <i>E. bieneusi</i> genotypes. In addition to <i>Cryptosporidium hominis</i> and <i>Cryptosporidium parvum</i>, two new pathogens identified in industrialized nations, <i>C. ubiquitum</i> and <i>Cryptosporidium viatorum</i>, were frequently detected. The two novel <i>C. ubiquitum</i> subtype families identified appeared to be genetic recombinants of known subtype families. Similarly, the dominant group 1 <i>E. bieneusi</i> genotypes and <i>G. duodenalis</i> subassemblage AII are known human pathogens. The similar distribution of human-pathogenic <i>Cryptosporidium</i> species and <i>E. bieneusi</i> and <i>G. duodenalis</i> genotypes between wastewater and CSO samples reaffirms that storm overflow is potentially a significant contamination source of pathogens in surface water. The frequent identification of <i>C. ubiquitum</i> and <i>C. viatorum</i> in urban wastewater suggests that these newly identified human pathogens may be endemic in China.<b>IMPORTANCE</b><i>Cryptosporidium</i> spp., <i>Giardia duodenalis</i>, and <i>Enterocytozoon bieneusi</i> are major waterborne pathogens. Their transport into surface water through combined sewer overflow, which remains largely untreated in developing countries, has not been examined. In addition, the identification of these pathogens to genotypes and subtypes in urban storm overflow and wastewater is necessary for rapid and accurate assessment of pathogen transmission in humans and transport in the environment. Data from this study suggest that, like untreated urban wastewater, combined sewer overflow is commonly contaminated with human-pathogenic <i>Cryptosporidium</i>, <i>G. duodenalis</i>, and <i>E. bieneusi</i> genotypes and subtypes, and urban storm overflow potentially plays a significant role in the contamination of drinking source water and recreational water with human pathogens. They also indicate that <i>Cryptosporidium ubiquitum</i> and <i>Cryptosporidium viatorum</i>, two newly identified human pathogens, may be common in China, and genetic recombination can lead to the emergence of novel <i>C. ubiquitum</i> subtype families.