Molecular epidemiology of human cryptosporidiosis in New South Wales, Australia

Cryptosporidium parasites have emerged as a significant threat to human and animal health. Initially considered as an opportunistic pathogen, Cryptosporidium is firmly established as one of the most common causes of global waterborne disease, capable of causing illness in both immune-compromised and immune-competent individuals. Essential to managing cryptosporidiosis and reducing risks of increasing prevalence is knowledge on the species contributing to disease and the potential zoonotic sources. The aim of this study was to investigate the molecular epidemiology of human cryptosporidiosis in New South Wales, Australia. Specifically, four aspects of cryptosporidiosis were investigated; the genetic diversity of Cryptosporidium causing disease in humans, the zoonotic role livestock and wildlife, the identification of demographic groups most at risk of disease and development of a molecular method that analyses parasite populations within the host. Molecular analyses of 447 human faecal samples, collected between January 2008 and December 2010, showed sporadic human cryptosporidiosis is caused by four species; C. hominis, C. parvum, C. andersoni and C. fayeri. Sequence analysis of the gp60 gene identified 5 subtype families and 31 subtypes. Cryptosporidium hominis IbA10G2 and C. parvum IIaA18G3R1 were the most frequent cause of sporadic disease, attributing to 59% and 16% of infections respectively. Between the months of January and April, 2009, NSW experienced the largest waterborne cryptosporidiosis outbreak reported in Australia to date. Molecular characterization identified the C. hominis IbA10G2 subtype as the causative parasite. In both sporadic and outbreak cases, an equal proportion of infections were found in males and females with cases most prevalent in the 0 - 4 year olds. The zoonotic role of livestock and wildlife was determined by screening faecal samples from cattle (n = 205) and eastern grey kangaroos (n = 76). Screening of cattle faecal samples identified four Cryptosporidium species; C. hominis, C. parvum, C. andersoni and C. ryanae. Subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, attributing to 47% of infections. Subtype IbA10G2 was also identified. Spatial analysis showed that cattle are an important zoonotic disease source. Cryptosporidium was not detected in kangaroo samples, indicating absence or low level infections. Molecular characterisation of the gp60 has facilitated contamination source tracking and increased understanding of the epidemiology of cryptosporidiosis. However the extent of genetic diversity observed, along with the exposure to the hosts' immune system, would support the hypotheses that significant selection pressure is placed on the gp60. A dual fluorescent terminal-restriction fragment length polymorphism analysis was developed to investigate the genetic diversity of Cryptosporidium subtype populations in a single host infection. This study showed subtype variation within infections and was the first to show that differences in the ratio of subtype populations occur between consecutive infections. Results from this thesis show human cryptosporidiosis is a complex relationship involving humans, animals and the environment, both inside and outside the host. Successful future management of cryptosporidiosis, particularly in Australia, will require a strong knowledgebase on the species present in livestock and native fauna and an understanding of the factors that influence contact between these sources and humans.