Subtyping Cryptosporidium ryanae: A Common Pathogen in Bovine Animals

Abstract

Cryptosporidium ryanae is one of the most common species for cryptosporidiosis in cattle. However, little is known of the genetic characteristics of C. ryanae due to the lack of subtyping tools. In the present study, the 60-kDa glycoprotein (gp60) gene of C. ryanae was identified in whole genome sequence data and analyzed for sequence characteristics using bioinformatics tools. The protein it encodes had some of the typical characteristics of GP60 proteins, with a signal peptide, a furin cleavage site, and a glycosylphosphatidylinositol anchor at the C terminus of the protein, and numerous O-glycosylation sites. The gene sequence was used in the development of a subtyping tool, which was used in characterizing C. ryanae from 110 specimens from dairy cattle, 2 from beef cattle, 6 from yaks, and 4 from water buffaloes in China. Altogether, 17 subtypes from 8 subtype families were recognized, namely XXIa to XXIh. Possible host adaption was identified within this species, reflected by the unique occurrence of XXIa, XXIc, and XXIh in dairy cattle, yaks, and water buffaloes, respectively. Some geographical differences were detected in the distribution of subtype families in dairy cattle; specimens from southern China showed higher genetic diversity than from northern China, and the XXIa subtype family was only seen in dairy cattle in southern and eastern China. The gp60-based subtyping tool should be useful in molecular epidemiological studies of the transmission of C. ryanae.

Keywords: 60-kDa glycoprotein; Cryptosporidium ryanae; geographical differences; host adaptation; subtyping tool.

Figure 1

Deduced amino acid sequence of the gp60 gene of Cryptosporidium ryanae (45,019) compared with sequences of C. parvum (AF022929), C. hominis (FJ839883), and C. ubiquitum [10]. Potential N-linked glycosylation sites are indicated in boldface and italic type, and predicted O-linked glycosylation sites are indicated in boldface and underlined type. The first 19 amino acids for the signal peptide and the last 17 amino acids for the GPI anchor are highlighted in green and blue, respectively. The classic furin proteolytic cleavage site sequence RSRR is highlighted in red. Dashes denote amino acid deletions.

Figure 2

Phylogeny among eight Cryptosporidium ryanae subtype families based on a maximum likelihood analysis of the partial gp60 gene. Sequences from dairy cattle, beef cattle, yaks, and water buffaloes are marked in black, green, red, and blue, respectively. Bootstrap values (>75) are indicated on branches. Scale bar indicates 0.02 nucleotide substitutions per site.

Figure 3

Deduced amino acid sequences of the partial gp60 gene of eight subtype families (XXIa to XXIh) in Cryptosporidium ryanae. Predicted O-linked glycosylation sites are indicated in boldface and underlined type. The classic furin proteolytic cleavage site sequence RSRR/RTRR is shown in red. Dashes denote amino acid deletions.