Classification of Cryptosporidium from sporadic cryptosporidiosis cases in humans employing sequence-based multilocus analysis following mutation scanning

Department of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia; Melbourne Diagnostic Unit, Victoria, 3052, Australia; Department of Epidemiology and Preventive Medicine, Monash University, Central and Eastern Clinical School, Alfred Hospital, Melbourne Victoria 3004, Australia; Melbourne Water Corporation, East Melbourne, Victoria 3002, Australia

^* To whom correspondence should be addressed. Email: a.jex{at}unimelb.edu.au.

	Abstract

In the present study, we analysed genetic variation among Cryptosporidium samples (n = 62) from humans with clinical cryptosporidiosis in South Australia. Sequence variation was assessed in regions within the small subunit of nuclear ribosomal RNA (pSSU), the 70 kilodalton heat shock protein (phsp70) and the 60 kilodalton glycoprotein (pgp60) genes employing single-strand conformation polymorphism (SSCP) analysis and sequencing. Based on the analyses of pSSU and phsp70, Cryptosporidium hominis (n = 38) and C. parvum (n = 24) were identified. The analysis of pgp60 revealed eight distinct subgenotypes classified as C. hominis IaA17R1 (n = 3), IbA9G3R2 (n = 14), IbA10G2R2 (n = 20) and IfA12G1R1 (n = 1) as well as C. parvum IIaA18G3R1 (n = 15), IIaA20G3R1 (n = 6), IIaA22G4R1 (n = 2) and IIcA5G3R2 (n = 1). Subgenotypes IaA17R1 and IIaA22G4R1 are new. Of the six other subgenotypes, IbA10G2R2, IIaA18G3R1, IIaA20G3R1 and IIcA5G3R2 were reported previously from the state of Victoria. This is the fourth record in Australia of C. parvum subgenotype IIaA18G3R1 from humans, which, to date, has only been isolated from cattle in other countries. This subgenotype might be a significant contributor to sporadic human cryptosporidiosis and may indicate a greater zoonotic contribution to infection of humans in the area of study. Comparative analyses revealed, for the first time, the differences in the genetic make-up of Cryptosporidium populations between two relatively close, major metropolitan cities.