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Cryptosporidium in fish: Morphological and molecular characterisation

Palermo, Cindy (2016) Cryptosporidium in fish: Morphological and molecular characterisation. Honours thesis, Murdoch University.

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Cryptosporidium is an Apicomplexa protozoan parasite that causes gastrointestinal illness in a wide range of vertebrate hosts, including humans. Little is known of the epidemiology of Cryptosporidium in fish. This study investigated the prevalence of Cryptosporidium in goldfish (Carassius auratus) (n=216) and mullet (Mugil cephalus) (n=13). Goldfish can be host to a range of Cryptosporidium sp. and research has shown that mullet has been host to genotype 3, therefore sampling from these breeds of fish could provide further characterisation. The fish were acquired from three sources in Metropolitan Perth, Western Australia; Vebas Aquarium (n=16), Water Garden Life fish farm (n=200) and a local bait shop in Fremantle (n=13). Intestines and stomachs were dissected and half kept for histology and the remaining half used for DNA extraction. All samples were initially screened at the 18S locus by quantitative PCR (qPCR) and positives further analysed by nested PCR and sequencing at the 18S and actin loci. Further subtyping was conducted on human-infectious species at the glycoprotein 60 (gp60) locus. The overall prevalence by qPCR was 30.1% (69/229) (CI 24.2-36.1). Of these only 34 samples amplified at the 18S locus and 23 clean sequences were obtained, with the remaining 11 sequences exhibiting mixed chromatograms. At the actin locus, 6 samples were successfully amplified and 3 clean chromatograms were obtained. Sequencing and phylogenetic analysis at the 18S locus identified C. parvum (n=2), C. hominis (n=10) and a novel species (n=11), which was identical to a novel genotype identified in a single isolate from a goldfish from a previous Honours project. Phylogenetic analysis confirmed that this novel genotype was genetically distinct and most closely related to C. scopthalmi (10.4% genetic distance). At the actin locus, all three isolates sequenced belonged to the novel genotype, which again grouped with C. scopthalmi and exhibited 14.1% genetic distance at this locus. Subtyping of C. hominis and C. parvum isolates at the gp60 locus was successful for 3 C. hominis isolates and all were typed as 1bA10G2, which is the main subtype involved in human outbreaks of cryptosporidiosis. Unfortunately, the parasite could not be identified in histological sections, which may be due to the patchy distribution of Cryptosporidium infections and rapid tissue autolysis. This is the first characterisation of the novel genotype at the actin locus and provides further support for its species status. The identification of human-infectious species in these fish is of public health concern as it may enable control of cryptosporidiosis outbreaks. Future research should focus on analysis of a wider range of fish species as well as clinical signs and histology to better understand the host range and pathogenicity of the novel genotype and the prevalence of human-infectious species in fish.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Ryan, Una, Paparini, Andrea, Kueh, Susan and Lymbery, Alan
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