Molecular epidemiology of waterborne zoonoses in the North Island of New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science (Epidemiology and Public Health) at Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North, New Zealand

Abstract

Campylobacter, Cryptosporidium and Giardia species are three important waterborne zoonotic pathogens of global public health concern. This PhD opens with an interpretive overview of the literature on Campylobacter, Cryptosporidium and Giardia spp. in ruminants and their presence in surface water (Chapter 1), followed by five epidemiological studies of Campylobacter, Cryptosporidium and Giardia spp. in cattle, sheep and aquatic environment in New Zealand (Chapters 2-6). The second chapter investigated four years of retrospective data on Campylobacter spp. (n=507) to infer the source, population structure and zoonotic potential of Campylobacter jejuni from six high-use recreational rivers in the Wanganui- Manawatu region of New Zealand through the generalised additive model, generalised linear/logistic regression model, and minimum spanning trees. This study highlights the ubiquitous presence of Campylobacter spp. in both low and high river flows, and during winter months. It also shows the presence of C. jejuni in 21% of samples containing highly diverse strains, the majority of which were associated with wild birds only. These wild birds-associated C. jejuni have not been detected in human, suggesting they may not be infectious to human. However, the presence of some poultry and ruminant-associated strains that are potentially zoonotic suggested the possibility of waterborne transmission of C. jejuni to the public. Good biosecurity measures and water treatment plants may be helpful in reducing the risk of waterborne Campylobacter transmission In the third study, a repeated cross-sectional study was conducted every month for four months to investigate the source of drinking source-water contamination. A total of 499 ruminant faecal samples and 24 river/stream water samples were collected from two rural town water catchments (Dannevirke and Shannon) in the Manawatu- Wanganui region of New Zealand, and molecular analysis of those samples was performed to determine the occurrence of Campylobacter, Cryptosporidium, and Giardia spp. and their zoonotic potential. The major pathogens found in faecal samples were Campylobacter (n=225 from 7/8 farms), followed by Giardia (n=151 from 8/8 farms), whereas Giardia cysts were found in many water samples (n=18), followed by Campylobacter (n=4). On the contrary, Cryptosporidium oocysts were only detected in a few faecal (n=18) and water (n=3) samples. Cryptosporidium and Giardia spp. were detected in a higher number of faecal samples from young animals (≤ 3 months) than juvenile and adult animals, whereas Campylobacter spp. were highly isolated in the faecal samples from juvenile and adult ruminants. PCRsequencing of the detected pathogens indicated the presence of potentially zoonotic C. jejuni and C. coli, Cryptosporidium parvum (gp60 allelic types IIA18G3R1 and IIA19G4R1) and Giardia duodenalis (assemblages AII, BII, BIII, and BIV) in cattle and sheep. In addition, potentially zoonotic C. jejuni and Giardia duodenalis assemblages AII, BI, BII, and BIV were also determined in water samples. These findings indicate that these three pathogens of public health significance are present in ruminant faecal samples of farms and in water, and may represent a possible source of human infection in New Zealand. In the fourth study, PCR-sequencing of Cryptosporidium spp. isolates obtained from the faeces of 6-week- old dairy calves (n=15) in the third study were investigated at multiple loci (18S SSU rDNA, HSP70, Actin and gp60) to determine the presence of mixed Cryptosporidium spp. infections. Cryptosporidium parvum (15/15), C. bovis (3/15) and C. andersoni (1/15), and two new genetic variants were determined along with molecular evidence of mixed infections in five specimens. Three main Cryptosporidium species of cattle, C. parvum, C. bovis and C. andersoni, were detected together in one specimen. Genetic evidence of the presence of C. Anderson and two new Cryptosporidium genetic variants are provided here for the first time in New Zealand. These findings provided additional evidence that describes Cryptosporidium parasites as genetically heterogeneous populations and highlighted the need for iterative genotyping at multiple loci to explore the genetic makeup of the isolates. The C. jejuni and C. coli isolates (n=96) obtained from cattle, sheep and water in the third study were subtyped to determine their genetic diversity and zoonotic potential using a modified, novel multi-locus sequence typing method (“massMLST”; Chapter 5). Primers were developed and optimised, PCR-based target-MLST alleles’ amplification were performed, followed by next generation sequencing on an Illumina MiSeq machine. A bioinformatics pipeline of the sequencing data was developed to define C. jejuni and C. coli multi-locus sequence types. This study demonstrated the utility and potential of this novel typing method, massMLST, as a strain typing method. In addition to identifying the possible C. jejuni/coli clonal complexes or sequence types of 68/96 isolates from ruminant faeces and water samples, this study reported three new C. jejuni strains in cattle in New Zealand, along with many strains, such as CC-61, CC-828 and CC-21, that have also been found in humans, indicating the public health significance of these isolates circulating on the farms in the two water catchment areas. Automation of the massMLST method and may allow a cost-effective high-resolution typing method in the near future for multilocus sequence typing of large collections of Campylobacter strains. In the final study (Chapter 6), a pilot metagenomic study was carried out to obtain a snapshot of the microbial ecology of surface water used in the two rural towns of New Zealand for drinking purposes, and to identify the zoonotic pathogens related to waterborne diseases. Fresh samples collected in 2011 and 2012, samples from the same time that were frozen, and samples that were kept in the preservative RNAlater were sequenced using whole-genome shotgun sequencing on an Illumina MiSeq machine. Proteobacteria was detected in all the samples characterised, although there were differences in the genus and species between the samples. The microbial diversity reported varied between the grab and stomacher methods, between samples collected in the year 2011 and 2012, and among the fresh, frozen and RNAlater preserved samples. This study also determined the presence of DNA of potentially zoonotic pathogens such as Cryptosporidium, Campylobacter and Mycobacterium spp. in water. Use of metagenomics could potentially be used to monitor the ecology of drinking water sources so that effective water treatment plans can be formulated, and for reducing the risk of waterborne zoonosis. As a whole, this PhD project provides new data on G. duodenalis assemblages in cattle, sheep and surface water, new information on mixed Cryptosporidium infections in calves, a novel “massMLST” method to subtype Campylobacter species, and shows the utility of shotgun metagenomic sequencing for drinking water monitoring. Results indicate that ruminants (cattle and sheep) in New Zealand shed potentially zoonotic pathogens in the environment and may contribute to the contamination of surface water. A better understanding of waterborne zoonotic transmission would help in devising appropriate control strategies, which could reduce the shedding of Campylobacter, Cryptosporidium, and Giardia spp. in the environment and thereby reduce waterborne transmission