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
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2016
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Massey University
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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.
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Keywords
Zoonoses, Epidemiology, Molecular epidemiology, North Island, New Zealand