Environmental Transport of Emerging Human-Pathogenic Cryptosporidium Species and Subtypes through Combined Sewer Overflow and Wastewater
Authors: Huang Chengchen , Hu Yue , Wang Lin , Wang Yuanfei , Li Na , Guo Yaqiong , Feng Yaoyu , Xiao Lihua 
Published: June 2017

The environmental transport of
Cryptosporidium
spp. through combined sewer overflow (CSO) and the occurrence of several emerging human-pathogenic
Cryptosporidium
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
Cryptosporidium
species (targeting the small subunit rRNA gene) and
Giardia duodenalis
(targeting the triosephosphate isomerase, β-giardin, and glutamate dehydrogenase genes) and
Enterocytozoon bieneusi
(targeting the ribosomal internal transcribed spacer) genotypes. Human-pathogenic
Cryptosporidium
species were further subtyped by sequence analysis of the 60-kDa glycoprotein gene, with additional multilocus sequence typing on the emerging zoonotic pathogen
Cryptosporidium ubiquitum. Cryptosporidium
spp.,
G. duodenalis
, and
E. bieneusi
were detected in 12 and 15, 33 and 32, and 37 and 40 CSO and wastewater samples, respectively, including 10
Cryptosporidium
species, 3
G. duodenalis
assemblages, and 8
E. bieneusi
genotypes. In addition to
Cryptosporidium hominis
and
Cryptosporidium parvum
, two new pathogens identified in industrialized nations,
C. ubiquitum
and
Cryptosporidium viatorum
, were frequently detected. The two novel
C. ubiquitum
subtype families identified appeared to be genetic recombinants of known subtype families. Similarly, the dominant group 1
E. bieneusi
genotypes and
G. duodenalis
subassemblage AII are known human pathogens. The similar distribution of human-pathogenic
Cryptosporidium
species and
E. bieneusi
and
G. duodenalis
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
C. ubiquitum
and
C. viatorum
in urban wastewater suggests that these newly identified human pathogens may be endemic in China.

IMPORTANCE
Cryptosporidium
spp.,
Giardia duodenalis
, and
Enterocytozoon bieneusi
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
Cryptosporidium
,
G. duodenalis
, and
E. bieneusi
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
Cryptosporidium ubiquitum
and
Cryptosporidium viatorum
, two newly identified human pathogens, may be common in China, and genetic recombination can lead to the emergence of novel
C. ubiquitum
subtype families.