Molecular-based investigation of Cryptosporidium and Giardia from animals in water catchments in southeastern Australia

There has been no large-scale systematic molecular epidemiological investigation of the waterborne protozoans, Cryptosporidium or Giardia, in southeastern Australia. Here, we explored, for the first time, the genetic composition of these genera in faecal samples from animals in nine Melbourne Water reservoir areas, collected over a period of two-years. We employed PCR-based single-strand conformation polymorphism (SSCP) and phylogenetic analyses of loci (pSSU and pgp60) in the small subunit (SSU) of ribosomal RNA and 60-kDa glycoprotein (gp60) genes to detect and characterise Cryptosporidium, and another locus (ptpi) in the triose-phosphate isomerase (tpi) gene to identify and characterise Giardia. Cryptosporidium was detected in 2.8% of the 2009 samples examined; the analysis of all amplicons defined 14 distinct sequence types for each of pSSU and pgp60, representing Cryptosporidium hominis (genotype Ib - subgenotype IbA10G2R2), Cryptosporidium parvum (genotype IIa - subgenotypes IIaA15G2R1, IIaA19G2R1, IIaA19G3R1, IIaA19G4R1, IIaA20G3R1, IIaA20G4R1, IIaA20G3R2 and IIaA21G3R1), Cryptosporidium cuniculus (genotype Vb - subgenotypes VbA22R4, VbA23R3, VbA24R3, VbA25R4 and VbA26R4), and Cryptosporidium canis, Cryptosporidium fayeri, Cryptosporidium macropodum and Cryptosporidium ubiquitum as well as six new pSSU sequence types. In addition, Giardia was identified in 3.4% of the samples; all 28 distinct ptpi sequence types defined were linked to assemblage A of Giardia duodenalis. Of all 56 sequence types characterised, eight and one have been recorded previously in Cryptosporidium and Giardia, respectively, from humans. In contrast, nothing is known about the zoonotic potential of 35 new genotypes of Cryptosporidium and Giardia recorded here for the first time. Future work aims to focus on estimating the prevalence of Cryptosporidium and Giardia genotypes in humans and a wide range of animals in Victoria and elsewhere in Australia. (Nucleotide sequences reported in this paper are available in the GenBank database under accession nos. KC282952-KC283005)