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.

FIG 1

Multiple alignment of amino acid (aa) sequences of species of Cryptosporidium for which complete gp60 ORFs were available, including C. viatorum. The signal peptide is indicated by an apostrophe, and amino acids coding for a transmembrane domain (GPI anchor) are indicated in italics. N-glycosylation sites are highlighted in black, and O-glycosylation sites are underlined. The furin cleavage site is highlighted in gray and reads “IVKR” in all of the C. viatorum isolates for which gp60 sequences are currently available (data not shown); on the other hand, note that a furin cleavage site for C. ubiquitum appears to be absent. Dashes (---) represent gaps, and asterisks (*) indicate residues identical in all sequences in the alignment. A colon (:) indicates that conserved (i.e., having similar characteristics) substitutions have been observed, and a period (.) indicates that semiconserved (i.e., having similar shape) substitutions have been observed.

FIG 2

Evolutionary relationships of 22 C. viatorum sequences (nearly complete ORFs). The evolutionary history was inferred using the neighbor-joining method (21). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method, and the unit of measure is the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2). The analysis involved 22-nucleotide sequences. The codon positions included were as follows: 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 858 positions in the final data set. Evolutionary analyses were conducted in MEGA6 (15). Bootstrap values lower than 50 have been removed.