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The role of dam water as a source of Cryptosporidium and Giardia infection in young sheep

Rawlings, Victoria (2018) The role of dam water as a source of Cryptosporidium and Giardia infection in young sheep. Honours thesis, Murdoch University.

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Cryptosporidium and Giardia are protozoan parasites that may cause gastrointestinal disease in a wide range of species, including humans and sheep, and impact profitability along the sheep meat supply chain. At present, sources of Cryptosporidium and Giardia infections on Australian sheep farms have not been described. This project aimed to determine whether contaminated drinking water supply, such as dams, may represent an important transmission source of Cryptosporidium and Giardia in extensively managed sheep, and whether the quantity of Cryptosporidium and Giardia (oo)cysts in dam water was greater during the winter months after high rainfall events. Water samples (n = 16) from 12 paddock dams and faecal samples (n = 274) from 11 mobs of sheep with access to the sampled dams were collected during autumn and winter from six commercial farms in south west Western Australia. Cryptosporidium oocysts and Giardia cysts were recovered from dam water samples using a calcium carbonate flocculation technique and genomic DNA was extracted from dam water and faecal samples with commercially available DNA extraction kits. Quantitative PCR (qPCR) was used to screen for the presence of Cryptosporidium spp. and Giardia spp. at the 18S ribosomal RNA (18S) locus and glutamate dehydrogenase (gdh) locus, respectively, and (oo)cyst quantitation was carried out using standards calibrated by droplet digital PCR (ddPCR) to estimate gene copy numbers. Conventional nested PCR was then used to amplify a longer fragment (>1 kb) of the 18S locus qPCR positive samples and the products were sequenced with Sanger sequencing to identify the Cryptosporidium species present in faecal and water samples. Cryptosporidium ubiquitum was further subtyped at the gp60 locus with nested PCR and Sanger sequencing. Conventional nested PCR that targeted the tpi locus of Giardia duodenalis assemblage A, B and E and Sanger sequencing were used on faecal samples that were qPCR positive for Giardia at the 18S locus to identify G. duodenalis assemblages. Conventional PCR was also performed on dam water samples. The overall prevalence of Cryptosporidium spp. in faecal samples was 25/274 (9.1%; 95% CI, 6.3-13.1) with C. xiaoi and C. ubiquitum subtype family XIIa detected. The overall prevalence of G. duodenalis with the gdh qPCR was 20/198 (10.1%; 95% CI, 6.6-15.1), with G. duodenalis assemblage A and E detected by nested PCR. There was no detection of G. duodenalis assemblage B in faecal or dam water samples. The average quantity of oocyst and cyst shedding in faeces was highest for C. ubiquitum (61,144 ± 97,999 (oo)cyst/g faeces) and lowest for G. duodenalis assemblage A (4,252 ± 3,081 (oo)cyst/g faeces). Cryptosporidium spp. was not detected in dam water samples with nested PCR. However, G. duodenalis assemblage A and E were detected by conventional nested PCR at the tpi locus, but Sanger sequencing only confirmed G. duodenalis assemblage E in 6/7 samples positive by nested PCR. G. duodenalis assemblage E was detected in 6/7 sheep flocks with access to dams positive for G. duodenalis assemblage E. There was no evidence of association between season and detection of pathogens in dam water. The findings from this project suggest that dam water may represent one source of transmission for G. duodenalis assemblage E infections in sheep, but other transmission routes are more likely for C. ubiquitum subtype XIIa, C. xiaoi and G. duodenalis assemblage A infections in these sheep. Future studies could aim to assess impact of other transmission routes, such as faecal contamination of pasture, feed or water accessed in the pre-weaning period.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Jacobson, Caroline, Zahedi, Alireza, Ryan, Una and Hancock, Serina
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