Molecular epidemiology of shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 STEC in calves in the North Island of New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand
Shiga toxin-producing Escherichia coli (STEC) are common inhabitants of the ruminant gastrointestinal
tract and have emerged as important zoonotic pathogens of global public health
concern. Four studies were carried out in the North Island of New Zealand to understand the
epidemiology of STEC O157 and non-O157 STEC in calves.
The aim of the first study was to determine population structure, transmission dynamics and
spatial relationship between genotypes of two zoonotic pathogens, STEC and Campylobacter
jejuni, found on farms in a defined catchment area in the Waikato region. Pooled faecal
samples (n=72) obtained from calves grazing in the catchment were analysed by RT-PCR for
E. coli O26, O103, O111, O145 and O157. The number of samples positive for O26 (30/72)
was high compared to O103 (7/72), O145 (3/72) and O157 (2/72) while no samples were
positive for O111. Using immuno-magnetic separation 18 O26, two O103 and a single O145
isolate were recovered from RT-PCR positive samples. Fifty-three C. jejuni isolates were recovered
from 72 pooled faecal samples. E. coli O26 and C. jejuni isolates were genotyped
using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing respectively.
All O26 isolates could be divided into three clusters on the basis of PFGE using XbaI. These
results indicated that E. coli O26 isolates recovered from calves on the same farm were generally
more similar than isolates recovered from different farms in the catchment. There were
a total of 13 different sequence types (STs) of C. jejuni isolated from the cattle and most
of the molecular variation (approximately 75%) resided between animals within farms. This
study suggested a high level of within farm transmission, and limited between-farm transmission
indicating direct contact between animals or sharing of personnel or equipment could be
important routes of transmission and good biosecurity measures may be helpful in reducing
The aim of the second study was to determine the distribution of virulence genes (stx1, stx2,
eae and ehxA) in E. coli isolates from dairy calves less than a week of age (bobby calves).
Sampling was carried out by systematic collection of recto-anal mucosal swab (RAMS) samples
from bobby calves slaughtered at two abattoirs in the North Island of New Zealand. The
samples were inoculated onto tryptone bile X-glucuronide (TBX) and sorbitol MacConkey
agar (CT-SMAC). Blue and white colonies (one each) and purple and grey colonies (one
each) were selected at random from TBX and CT-SMAC plates respectively. In total 975 E.
coli isolates obtained from the two media were analysed by multiplex PCR to detect stx1,
stx2, eae and ehxA genes. The most common combination of virulence markers were eae,
ehxA (n=35) followed by eae (n=9). Only eight STEC were identified of which four were
stx2, eae, ehxA-positive (3 O157:H7 and 1 O68:H24), three were stx1, eae, ehxA (2 O26 and
1 O71:HR) and there was one stx2-only isolate (ONT:HNM). All the isolates could be divided
into 15 clusters with >70% similarity using XbaI by PFGE. These findings indicate
that STEC of public health significance such as O157 and O26 are present in bobby calves
and may represent an important source of human infection in New Zealand.
In the third study samples obtained in the second study were processed to determine the
occurrence and spatial distribution of E. coli O26, O103, O111 and O145 in bobby calves
in the North Island of New Zealand. The association of IgG concentration, weight, sex and
breed with occurrence of O26, O103, O111 and O145 as determined by direct RT-PCR on
RAMS enrichments was also investigated. Using RT-PCR 134/299 (44.8%) of RAMS were
positive for O26, 68/299 (22.7%) for O103 and 47/299 (15.7%) for O145. No RAMS samples
were RT-PCR positive with O111-specific primers. The success of isolation by culture of E.
coli O26 (49/134 isolates) was higher from RT-PCR positive samples as compared to O103
(4/68 isolates) and O145 (5/47 isolates). Using a logistic regression model no association was
observed between PCR prevalence and the variables IgG, sex or breed of the calves. However,
calves positive for O26 were more likely to be positive for O103 and vice versa (P=0.01) and
similar association was found between calves positive for O145 and O103 (P=0.03). O26
isolates could be grouped into four clusters (A-D) of >70% similarity using XbaI PFGE. K
function analysis did not indicate any evidence of spatial clustering of farms positive for O26,
O103 or O145. This study indicted that O26 is more prevalent in bobby calves as compared
to O103 and O145 and colostrum feeding may not be helpful in reducing the carriage of E.
coli O26, O103 and O145.
In the fourth study E. coli isolates (n=137) obtained from previous studies were genotyped using
PFGE and allelic profiling (based on 8 genes). The eae and ehxA genes of E. coli isolates
were subtyped using PCR-restriction fragment length polymorphism (RFLP) analysis. Endonuclease
digestion of ehxA PCR products with TaqI from 121 ehxA positive E. coli isolates
resulted in six ehxA subtypes (A-F). Endonuclease digestion with HhaI or HaeIII enzymes of
the 129 eae positive isolates indicated that 82 were followed by (n=34),
(n=11) and /
(n=2). An association between eae subtype and E. coli serogroup was also observed. All O26
isolates were subtype , all O103 isolates were subtype and all O157 isolates were
. E. coli
isolates were also analysed for plasmid-associated allels espP, etpD, katP and -hly genes. Of
137 isolates 93 (67.8%) were positive for espP, 32 for etpD (23.3%), 76 for katP (55.4%) and
nine for the -hly (6.5%) gene. The genotyping ability of allelic profiling was compared with
PFGE profiling using PERMANOVA and multidimensional scaling and the results indicated
that isolates having similar allelic profiles had similar PFGE profiles and tended to cluster
together. The results also indicted that eae and ehxA profiles could explain a high population
of the variance in PFGE profiles. These results may provide basis for the development of new
genotyping method for E. coli isolates, having high discriminatory power and being easier
to perform. The E. coli serogroups and eae and ehxA subtypes observed in these serogroups
have also been observed from human E. coli isolates indicating the public health significance
of these isolates.
These studies provide useful information about epidemiology of O157 and non-O157 in calves
in the North Island of New Zealand and indicate that calves may be an asymptomatic reservoir
of STEC and a possible source of infection for humans. Better understanding of the population
structure and transmission of STEC would help in devising appropriate control strategies. The
implementation of these control measures could reduce the prevalence of STEC in calves and
thus reduce transmission to humans.