Development of a gp60-subtyping method for Cryptosporidium felis

Abstract

Background: Feline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70.

Methods: We have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker.

Results: Sequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed.

Conclusions: We have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations.

Keywords: 60-kDa glycoprotein; Cryptosporidiosis; Epidemiological marker; Genetic variability; Molecular typing; Source tracking; Zoonotic transmission.

Fig. 1

Features of the gp60 gene of C. felis. Amino acid alignment illustrating the differences in the gp60 between Cryptosporidium species. The insertion present in C. felis is highlighted in blue and the serine repeats region characteristic of other Cryptosporidium species is highlighted in green. Cryptosporidium ubiquitum lacks the typical S repeat, the region is highlighted in yellow

Fig. 2

Phylogenetic analysis of 102 gp60 sequences from C. felis isolated from humans and cats. Blue dots indicate sample from human and green squares indicate sample from cat. All cases with known travel history have been indicated while samples with unknown travel history are marked with an asterisk

Fig. 3

a In zoonotic case 2 (Z2) one nucleotide in the gp60 sequence clearly showed how a dominant variant of C. felis shifted to another when transmitted from one host (cat) to another (human). The nucleotide in position 833 differed between the cat (G) and the owner (A). Massive parallel sequencing of the cat sample, covering the named position 62 times, showed that one of the 62 reads indeed had the adenine (A) found in the human sample. b All other cats and humans in the study had A in this specific position