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.

Subtype distribution of zoonotic pathogen Cryptosporidium felis in humans and animals in several countries

“…In the maximum likelihood tree, the 44 DNA sequences formed four distinct clusters as expected, with 99-100% bootstrap support (Figure 1). Three of the four clusters were found in the previous study [10], while one cluster seen in the previous study, B, was absent from the present study. In accordance with the established nomenclature system for Cryptosporidium subtype families [11], the four clusters found in the present study were named as XIXa, XIXc, XIXd and XIXe (newly identified in the present study) subtype families, while the previous Cluster B was named as XIXb subtype family.…”

“…Among them, C. felis was initially reported in domestic cats from Japan in 1979 and proposed as a new species based on host occurrence and oocyst morphology, which was later supported by genetic characterizations of isolates [6,7]. This species has been reported in numerous human cases subsequently [8], and sometimes in association with the presence of infection in cats in the same household [9,10].…”

“…A gp60-based subtyping tool has become available recently for C. felis [10]. Thus far, the genetic characterization of C. felis has been mostly restricted to isolates acquired in three European countries (the United Kingdom, Sweden and Denmark).…”

Subtyping Cryptosporidium ryanae: A Common Pathogen in Bovine Animals

“…This sequence difference is probably responsible for the failure of PCR to amply the gp60 locus in C. ryanae using the universal primers designed based on the sequences of C. parvum and C. hominis [21]. Unlike the gp60 gene of C. parvum and other closely related species/genotypes, the TCA/TCG/TCT repeats, which encode a polyserine tract and are used in differentiating subtypes within subtype families, are absent in the gp60 gene of C. ryanae, C. ubiquitum, and C. felis [5,10,23]. In C. ryanae, there is a polyglycine tract encoded by GTT repeats in this region of the gp60 gene.…”

“…The application of gp60 subtyping tools has contributed greatly to our understanding of the genetic diversity, transmission dynamics, as well as host adaption in human-pathogenic Cryptosporidium spp. [5][6][7][8][9][10][11]. There is also a subtyping tool for C. andersoni based on sequence analysis of several genetic loci with simple tandem repeats [12].…”