High applicability of a novel method for gp60-based subtyping of Cryptosporidium meleagridis

Abstract

Cryptosporidium meleagridis is a common cause of cryptosporidiosis in avian hosts and the third most common species involved in human cryptosporidiosis. Sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) gene is a frequently used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium. However, few studies have included gp60 sequencing of C. meleagridis. One explanation may be that the gp60 primers currently in use are based on Cryptosporidium hominis and Cryptosporidium parvum sequence data, potentially limiting successful amplification of the C. meleagridis gp60 gene. We therefore aimed to design primers for better gp60 subtyping of C. meleagridis. Initially, ∼1,440 bp of the gp60 locus of seven C. meleagridis isolates were amplified using primers flanking the open reading frame. The obtained sequence data (∼1,250 bp) were used to design primers for a nested PCR targeting C. meleagridis. Twenty isolates (16 from human and 4 from poultry) previously identified as C. meleagridis by analysis of small subunit (SSU) rRNA genes were investigated. Amplicons of the expected size (∼900 bp) were obtained from all 20 isolates. The subsequent sequence analysis identified 3 subtype families and 10 different subtypes. The most common subtype family, IIIb, was identified in 12 isolates, represented by 6 subtypes, 4 new and 2 previously reported. Subtype family IIIe was found in 3 isolates represented by 3 novel, distinct subtypes. Finally, IIIgA31G3R1 was found in 1 human isolate and 4 poultry isolates, all originating from a previously reported C. meleagridis outbreak at a Swedish organic farm.