Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum

Abstract

The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species-or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles. Cryptosporidium is one of the leading causes of infant and childhood morbidity (1). Nearly 20 species and genotypes of Cryptosporidium have been isolated from human stool (2). While Cryptosporidium parvum and Cryptosporidium hominis account for the majority of human cases of cryptosporidiosis, many other species and genotypes, most of which are acknowledged as zoonotic, appear to occur sporadically or rarely in outbreaks all over the world (3-7). The availability of relevant, precise, and robust typing tools is therefore critical to outbreak investigation, surveillance, and control of the parasite.Molecular characterization of Cryptosporidium based on gp60 gene data has proved to have high epidemiological relevance (8).To date, the resolution of the gp60 locus has enabled a subtle classification of species and genotypes into subtype families, which in turn can be differentiated at the subtype level. However, the high resolution and genetic variability existing in the gp60 locus also entails a challenge in terms of developing primers applicable to the many species and genotypes of Cryptosporidium (4, 9-11). The primers commonly used for gp60 subtyping of C. parvum and C. hominis (12) do not reliably amplify many other Cryptosporidium species. For example, new primers have been published for Cryptosporidium meleagridis (9) and Cryptosporidium ubiquitum (11). Similarly, the C. parvum/C. hominis primers do not apply to C. viatorum (4, 10), one of the panoply of Cryptosporidium species infecting humans and first described in 2012 (10); the 10 cases identified in the United Kingdom at that point all had a history of recent return from traveling to the Indian subcontinent. Later, C. viatorum was identified in patients in Nigeria (13) and Ethiopia (14) and in Swedish patients who had travelled to Kenya or Guatemala (4, 5).The aims of the present study were, first, to amplify, sequence, and characterize the gp60 gene of C. viatorum, and second, to develop, validate, and apply primers appropriate for gp60 typing of C. viatorum, thus complementing the panel of Cryptosporidium gp60 primers and contributing to C. viatorum gp60 data useful for epidemiologica...

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

“…The two C. ubiquitum subtypes found in this study belong to two novel subtype families (21), and the C. viatorum subtype (XVaA6) found in this study has not been reported elsewhere (8). In phylogenetic analysis of gp60 sequences, the new C. ubiquitum subtype families formed a cluster with the rodent subtype families XIIb, XIIc, and XIId rather than the ruminant subtype family XIIa.…”

“…However, it is becoming an important Cryptosporidium species in humans in industrialized nations (21,22), although it has been reported in only a few human cases in developing countries (21,23,24). In contrast, C. viatorum is known as a human pathogen in developing countries, as human infections in the United Kingdom and Sweden have been linked to travels to India, Pakistan, Bangladesh, Kenya, Barbados, and Guatemala (8). However, its presence in developing countries has been confirmed only in Ethiopia and Nigeria (25,26).…”

“…Similarly, one C. viatorum-positive wastewater sample was identified as a new subtype, XVaA6, according to the established nomenclature (8).…”

“…Specimens that were positive for C. hominis or C. parvum were subtyped by nested PCR analysis of the 60-kDa glycoprotein (gp60) gene (7). Those containing C. meleagridis, C. ubiquitum, and C. viatorum were subtyped at the gp60 locus using PCR methods developed specifically for these human pathogens (8,21,29). C. ubiquitum was further characterized by a newly established MLST method (27).…”

Subtype analysis of zoonotic pathogen Cryptosporidium skunk genotype

“…One common target used in subtyping of these species is the 60-kDa glycoprotein (gp60) gene, as it is highly polymorphic and divides the two Cryptosporidium species into several major subtype families (Strong et al, 2000). In recent years, subtyping tools based on gp60 sequences have been developed for other Cryptosporidium species and genotypes such as C. meleagridis , C. ubiquitum , C. fayeri , C. viatorum , and Cryptosporidium chipmunk genotype I (Guo et al, 2015; Li et al, 2014; Power et al, 2009; Stensvold et al, 2014; Stensvold et al, 2015). With the availability of whole genome sequences, some of the new tools, such as the one for Cryptosporidium chipmunk genotype I, have used conserved nucleotide sequences in PCR primer design (Guo et al, 2015).…”