Wild rats (Rattus spp.) carry many zoonotic pathogens including Cryptosporidium. Due to the close proximity of rats to humans in urban environments, the potential for disease transmission is high. Cryptosporidium is a protozoan parasite which when ingested causes serious human illness. Despite its importance, genetic characterization of Cryptosporidium in wild rats in the Hainan province of China has not been performed. In this study, we analyzed the occurrence and genetics of Cryptosporidium in wild rats from Hainan, China. From December 2017 to October 2018, 150 wild rats were captured and fresh fecal material was collected from intestinal sections. Rat species were identified by PCR-based amplification and analysis of the vertebrate cytochrome b (cytb) gene. Cryptosporidium was examined by PCR amplification of the partial small subunit of ribosomal DNA (SSU rDNA). C. viatorum were subtyped by PCR analysis of the gp60 gene. A total of four rat species were identified including Asian house rats (Rattus tanezumi) (n = 46), brown rats (Rattus norvegicus) (n = 56), Edward's long-tailed rats (Leopoldamys edwardsi) (n = 38) and muridae (Niviventer fulvescens) (n = 10), with Cryptosporidium positive rates of 73.9%, 28.6%, 55.3% and 40.0%, respectively (average infection rate: 50.0%, 75/150. Sequence analysis confirmed the presence of four Cryptosporidium species and two genotypes including C. viatorum (n = 11); C. occultus (n = 2); C. muris (n = 1); and C. erinacei (n = 1); rat genotypes III (n = 13) and IV (n = 47). Three novel subtypes of C. viatorum were identified in 6 of the 11 infected Edward's long-tailed rats: XVcA2G1a (n = 4), XVcA2G1b (n = 1) and XVdA3 (n = 1). The identification of human pathogenic C. viatorum and zoonotic C. occultus, C. muris and C. erinacei, suggested that wild rats infected with Cryptosporidium pose a threat to human health. Taken together, these findings highlight the need to control the rat population in Hainan, China. The need to improve the public awareness of the risk of disease transmission from wild rats to humans is also highlighted.

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