Cameron Gordon

Specific and quantitative detection and identification of Cryptosporidium hominis and C. parvum in clinical and environmental samples.

Cryptosporidium is an enteric protozoan parasite that is resistant to inactivation by commonly used drinking water disinfectants. Between 2004 and 2010, it was responsible for 60% of all waterborne protozoan parasitic outbreaks reported worldwide. Most sporadic infections in humans and almost all outbreaks are caused by Cryptosporidium parvum and Cryptosporidium hominis. We report the development and validation of a quantitative qPCR assay using minor groove binder (MGB)-probes targeting a unique Cryptosporidium specific protein-coding gene, that directly detects, quantitates and identifies C. hominis and C. parvum in environmental and 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 cross-reactions 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%, respectively 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 18S locus. 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.

Prevalence and molecular characterisation of Cryptosporidium and Giardia species in pre-weaned sheep in Australia

A total of 477 faecal samples from pre-weaned sheep from 5 different farms in the south west of Western Australia were screened for the presence of Cryptosporidium and Giardia using PCR. There were substantial differences in prevalence between the farms and overall prevalence was 24.5% and 11.1%, respectively for Cryptosporidium and Giardia. At the 18S locus, 66 Cryptosporidium positives were identified, the majority of which were C. bovis (n=52), followed by the cervid genotype (n=10) and C. parvum (n=2). At a second diagnostic locus, using C. parvum and C. hominis-specific qPCR primers, 63 C. parvum positives were identified, some of which were co-infections with C. bovis. The C. parvum/C. hominis qPCR was more sensitive than the nested 18S PCR at detecting C. parvum. This may be due to the low numbers of oocysts present, as quantitation data indicated that all the C. parvum detected were present in low numbers (1-10 oocysts). It may also be that using C. parvum-specific primers is necessary to determine the true prevalence of C. parvum. Amongst Giardia positive isolates, G. duodenalis genotype E (livestock) was the most prevalent (36/53), with G. duodenalis genotype A detected in five positive isolates. There were also 11 mixed A and E infections detected. The findings of the present study indicate that pre-weaned lambs are not an important source of zoonotic Giardia genotypes in Australia but may be an important source of zoonotic Cryptosporidium.

Molecular characterization of Cryptosporidium and Giardia in pre-weaned calves in Western Australia and New South Wales.

A total of 364 fecal specimens from randomly selected pre-weaned calves, aged up to 4 months, from 5 different farms in the south of Western Australia and 1 farm from New South Wales were screened for the presence of Cryptosporidium and Giardia using PCR. There were substantial differences in prevalence between the farms and the overall prevalence was 22.3% (81/364) and 26.9% (98/364) respectively for Cryptosporidium and Giardia. For Cryptosporidium, 70 positives were identified at the 18S locus. At a unique diagnostic locus, an additional 12 C. parvum positives were identified. Sequence analysis at the 18S ribosomal RNA locus was successful for 59 of the 70 positive isolates; of these 14 were C. parvum, 28 were C. bovis, 15 were C. ryanae, 1 was pig genotype II and 1 was a mixed C. ryanae/C. parvum infection. Sub-typing analysis at the glycoprotein 60 (gp60) locus for 24 C. parvum isolates identified all as IIa; 17 were A17G2R1, 1 was A18G3R1 and 6 were A20G3R1. For Giardia, 75 positives were identified at the 18S locus and an additional 23 positives were identified at the gdh locus. The majority of the isolates sequenced were assemblage E, however assemblage A and B and mixed A and E and A, B and E infections as well as the quenda genotype were identified. The findings of the present study indicate that pre-weaned calves are not an important source of zoonotic Giardia species in Australia but may be an important source of zoonotic Cryptosporidium.

Prevalence of Cryptosporidium species in recreational versus non-recreational water sources

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, represents the major public health concern of water utilities in developed nations due to its small size, resistance to disinfection and ability to be shed in large numbers in faeces. In Australia, recreational access is not allowed on direct supply sources, however, in Western Australia, limited recreational access to drinking water catchments has been allowed, although only in the outer catchment. Recreational activities within 2 km of the drinking water body is prohibited. The present study compared the amount, prevalence and species of Cryptosporidium in recreational versus non-recreational water catchments in the south west of Western Australia (WA). Recreational water catchments, which allowed swimming and camping had a higher prevalence of Cryptosporidium and the majority of samples were the human-associated C. hominis. Non-recreational catchments had a lower prevalence and all the samples genotyped were C. parvum. Risk analysis identified increasing population as strongly correlated with an increase in the prevalence of Cryptosporidium in recreational catchments. This suggests that recreational access to drinking water catchments is a serious public health risk and government policy limiting activities to the outer catchment should be supported.

Novel primer sets for next generation sequencing-based analyses of water quality

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.