This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. faecal samples. An internal amplification control (IAC) was also developed and included in this assay. The qPCR assay was compared with an 18S nested PCR assay for sensitivity and specificity. The analytical sensitivity for the qPCR assay was 1 oocyst and 1-10 oocysts for the 18S assay. Evaluation of analytical specificity of the qPCR assay revealed no crossreactions with other genera and detected all C. parvum and C. hominis isolates correctly. The diagnostic sensitivity and specificity of the qPCR was 100% compared to 96.9% and 98.4% Specific and quantitative detection and identification ofrespectively for the 18S assay. The qPCR assay was also highly reproducible with RSD (relative standard deviation) values of 1.4-9.4%, when the assay was performed by four different technicians. When tested on water samples, the qPCR assay was more sensitive than the 18S assay, detecting positives in 37 of 138 water samples compared to 35 for the 18Slocus. This qPCR assay should be a valuable tool for the detection and differentiation of C.hominis and C. parvum in both clinical and environmental samples.

As part of long-term monitoring of Cryptosporidium in water catchments serving Western Australia, New South Wales (Sydney) and Queensland, Australia, we characterised Cryptosporidium in a total of 5774 faecal samples from 17 known host species and 7 unknown bird samples, in 11 water catchment areas over a period of 30 months (July 2013 to December 2015). All samples were initially screened for Cryptosporidium spp. at the 18S rRNA locus using a quantitative PCR (qPCR). Positives samples were then typed by sequence analysis of an 825 bp fragment of the 18S gene and subtyped at the glycoprotein 60 (gp60) locus (832 bp). The overall prevalence of Cryptosporidium across the various hosts sampled was 18.3% (1054/5774; 95% CI, 17.3-19.3). Of these, 873 samples produced clean Sanger sequencing chromatograms, and the remaining 181 samples, which initially produced chromatograms suggesting the presence of multiple different sequences, were re-analysed by Next- Generation Sequencing (NGS) to resolve the presence of Cryptosporidium and the species composition of potential mixed infections. The overall prevalence of confirmed mixed infection was 1.7% (98/5774), and in the remaining 83 samples, NGS only detected one species of Cryptosporidium. Of the 17 Cryptosporidium species and four genotypes detected (Sanger sequencing combined with NGS), 13 are capable of infecting humans; C. parvum, C. hominis, C. ubiquitum, C. cuniculus, C. meleagridis, C. canis, C. felis, C. muris, C. suis, C. scrofarum, C. bovis, C. erinacei and C. fayeri. Oocyst numbers per gram of faeces (g) were also determined using qPCR, with medians varying from 6021-61,064 across the three states. The significant findings were the detection of C. hominis in cattle and kangaroo faeces and the high prevalence of C. parvum in cattle. In addition, two novel C. fayeri subtypes (IVaA11G3T1 and IVgA10G1T1R1) and one novel C. meleagridis subtype (IIIeA18G2R1) were identified. This is also the first report of C. erinacei in Australia. Future work to monitor the prevalence of Cryptosporidium species and subtypes in animals in these catchments is warranted.

22Reliable identification of cyanobacterial isolates has significant 23 socio-economic implications as many bloom-forming species 24 affect the aesthetics and safety of drinking water, through the 25 production of taste and odour compounds or toxic metabolites. 26The limitations of morphological identification have promoted 27 the application of molecular tools, and encouraged the adoption 28 of combined (polyphasic) approaches that include both 29 microscopy-and DNA-based analyses. In this context, the 30 rapid expansion of available sequence data is expected to allow 31 increasingly reliable identification of cyanobacteria, and 32 ultimately resolve current discrepancies between the two 33 approaches. 34In the present study morphological and molecular 35 characterisations of cyanobacterial isolates (n=39), collected 36 from various freshwater sites in Australia, were compared. 37Sequences were obtained for the small ribosomal subunit RNA 38 gene (16S rDNA) (n=36), the DNA-dependent RNA 39 polymerase gene (rpoC1) (n=22), and the phycocyanin operon, 40 with its intergenic spacer region (cpcBA-IGS) (n= 19).

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Naegleria fowleri is a free-living amoeboflagellate inhabiting soil and water that can cause Primary Amoebic Meningoencephalitis (PAM), a rare and sometimes fatal disease. In Australia, the amoeba typically inhabits drinking water supplies that have consistent water temperatures above 20°C. The incidence of PAM is widespread in Australia, with reports from South Australia, Western Australia, New South Wales and Queensland. One of the key issues for water utilities is the potential widespread distribution of N. fowleri and its ability to infect and re-infect drinking water supplies. In Western Australia, the majority of drinking water supplies are operated by the Water Corporation. This paper describes the conditions under which Naegleria spp. have been detected and describes the operational methods employed by the Water Corporation to control and mitigate Naegleria in public drinking water supplies.