Molecular Epidemiology of Campylobacter jejuni Isolates from Wild-Bird Fecal Material in Children's Playgrounds䌤
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 779–783 Vol. 75, No. 3
0099-2240/09/$08.00⫹0 doi:10.1128/AEM.01979-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Molecular Epidemiology of Campylobacter jejuni Isolates from
Wild-Bird Fecal Material in Children’s Playgrounds䌤
Nigel P. French,1* Anne Midwinter,1 Barbara Holland,2 Julie Collins-Emerson,1 Rebecca Pattison,1
Frances Colles,3 and Philip Carter4
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand1; Institute of
Fundamental Sciences, Massey University, Palmerston North, New Zealand2; The Peter Medawar Building and Department of
Zoology, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3SY3; and Institute for
Environmental Science and Research, Kenepuru Science Centre, Porirua, New Zealand4
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Received 26 August 2008/Accepted 23 November 2008
In many countries relatively high notification rates of campylobacteriosis are observed in children under 5
years of age. Few studies have considered the role that environmental exposure plays in the epidemiology of
these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young
children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We
investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192
samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp.
Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus
sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was
isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177;
all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in
profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the
ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these
birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild
birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further,
the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations
in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds
in the 19th century.
Campylobacteriosis is a common disease of humans world- highest in children between 1 and 4 years old (578/100,000) (2).
wide and a major burden on the health service in many nations. The potential environmental pathways for infection in this
In New Zealand the reported rate of campylobacteriosis is one preschool age group include exposure to wild-bird feces in
of the highest in the world, and a recent estimate is that there open playgrounds. Many playgrounds are located in parks that
are around 300 cases per 100,000 people per year (2). Despite are natural habitats for wild birds; play equipment provides
many years of concerted research effort, many questions re- ideal perching sites for wild birds, and overhanging trees pro-
lated to the complex epidemiology and control of this disease vide ideal roosting areas. Children’s behavior, particularly the
remain unanswered. Much emphasis has been placed on con- frequent hand-mouth contact in this age group, is also likely to
trolling exposure pathways involving food, particularly poultry increase the risk of ingesting infective material (3, 13, 25).
(1). This can be justified due to the high carriage rates in In this study we investigated the possibility that wild-bird
poultry (26) and due to evidence that similar strains are
contamination of children’s playgrounds is a source of human
present on poultry and in human infections (26).
infection by estimating the prevalence of Campylobacter jejuni
Other sources, both food and environmental, are also con-
in fresh and dried wild-bird fecal samples and comparing the
sidered important contributors to human infection (7, 12, 19,
20). It is likely that there are multiple diverse other pathways genotypes isolated from these samples with human clinical
and that further public health gains beyond those achieved by isolates. As far as we are aware, this is the first study in New
the control of poultry contamination will be possible only Zealand, and possibly elsewhere, to isolate Campylobacter spp.
through systematic identification and control of these alterna- from children’s playgrounds and to examine the potential zoo-
tive pathways. These pathways include cattle and sheep meat, notic risk from these organisms, and it is a unique use of
occupational exposure, and direct contact and contamination molecular epidemiology for investigation of a potential envi-
of the environment with wildlife and livestock fecal material. ronmental risk pathway. This study also provided the first op-
In New Zealand the reported rate of campylobacteriosis is portunity to compare wild-bird genotypes obtained from an
urban area in New Zealand with wild-bird genotypes from
other countries recorded in the Campylobacter PubMLST da-
* Corresponding author. Mailing address: Institute of Veterinary, tabase (http://pubmlst.org/campylobacter/). This is particularly
Animal and Biomedical Sciences, Massey University, Palmerston
relevant given the historical transfer of urban-dwelling birds
North, New Zealand. Phone: 64 (06) 350 5078. Fax: 64 (06) 350 5714.
E-mail: N.P.French@massey.ac.nz. from the United Kingdom to New Zealand in the 19th century
䌤
Published ahead of print on 1 December 2008. (24).
779780 FRENCH ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 1. Multilocus sequence types of C. jejuni isolates from wild-bird fecal material contaminating children’s playgrounds in New Zealand
No. of Allelic nucleotide sequence no.d
Sequence type Clonal complex
isolates aspA glnA gltA glyA pgm tkt uncA
12 4 7 10 4 1 7 1 45a ST-45b
1 4 7 10 4 42 7 1 137a ST-45b
1 4 7 10 4 42 51 1 583a ST-45b
1 35 43 9 5 8 46 21 681a ST-682b
1 99 128 91 125 170 146 111 1324a Uc
1 37 4 4 48 13 25 23 2354a U
2 35 185 162 5 8 222 21 2536 U
1 17 2 8 5 8 2 143 2537 ST-177b
1 35 2 8 51 361 2 21 2538 U
1 17 2 9 5 8 222 21 2539 ST-177b
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a
Sequence type previously associated with wild birds.
b
Clonal complex previously associated with wild birds.
c
U, unassigned.
d
Allele nucleotide sequence numbers in the PubMLST database (http://pubmlst.org/campylobacter).
MATERIALS AND METHODS represent the same split. The edge lengths are drawn proportional to the mean
length of the edge in all the trees where it appears.
Samples were obtained from all 10 publicly accessible children’s playgrounds
in the southeastern sector of the city of Palmerston North, New Zealand, be-
tween 8 November 2004 and 2 February 2005. The maximum distance between
playgrounds was 3 km. RESULTS
Isolation of Campylobacter spp. Bird fecal samples were collected using sterile
cotton-tipped swabs that were immediately placed into 3 ml of Bolton broth (Lab Contamination of playgrounds with bird feces. A total of
M, Bury, England). The broth was incubated at 42°C in a microaerobic atmo- 24/192 samples (12.5%) were positive for C. jejuni, including
sphere (85% N2, 10% CO2, 5% O2) for 48 h before an aliquot was plated onto
4/60 dried samples (6.7%) and 20/132 fresh samples (15.2%).
mCCDA agar (Fort Richard, Auckland, New Zealand). Plates were examined
after 48 h of incubation at 42°C in a microaerobic atmosphere. Isolates were Positive samples were found on wood, concrete, soil, bark,
examined by dark-field microscopy to determine morphology and motility and plastic, and grass surfaces. The predominant species of birds
tested to determine whether oxidase was produced. The isolates that had typical observed in the playgrounds at the time of sampling were the
morphology and motility and for which the oxidase test was positive were frozen European starling (Sturnus vulgaris), house sparrow (Passer
at ⫺80°C in 15% glycerol broth (Oxoid, Basingstoke, England). Isolates were
identified as C. jejuni by PCR (23).
domesticus), welcome swallow (Hirundo tahitica), blackbird
MLST. Multilocus sequence typing (MLST) was performed as described by (Turdus merula), Australian magpie (Gymnorhina tibicen),
Dingle et al. (10). Chromosomal DNA was prepared from freshly grown cultures song thrush (Turdus philomelos), tui (Prosthemadera novaesee-
by boiling the cultures for 10 min, followed by centrifugation of the disrupted landiae), chaffinch (Fringilla coelebs), and goldfinch (Carduelis
cells. The supernatant was decanted into a fresh tube and used for amplification.
carduelis).
Amplification was performed in a 25-l reaction mixture using Applied Biosys-
tems AmpliTaq Gold master mixture (Applied Biosystems, Auckland New Zea- Molecular typing. Twenty-two isolates were typed by MLST.
land) and 5 pmol of each primer. Products were sequenced with an ABI 3130XL Two other isolates could not be resuscitated from frozen cul-
automated DNA sequencer using ABI BigDye v3.1 (Applied Biosystems) ac- ture for further characterization. Sequence type 45, the
cording to the manufacturer’s instructions. Sequence data were collated, and founder sequence type of clonal complex ST-45, was the pre-
alleles were assigned using the Campylobacter PubMLST database (http:
//pubmlst.org/campylobacter/). Novel alleles and sequence types were submitted
dominant type, accounting for 12/22 (54.5%) isolates. Two
for allele and ST designations when appropriate. other isolates, designated ST-137 and ST-583, also belonged to
PFGE. Isolates were characterized further using the pulsed-field gel electro- clonal complex ST-45 (Table 1). The remaining eight isolates
phoresis (PFGE) method of Ribot et al. (21), and the profiles were compared either belonged to clonal complexes ST-177 or ST-682 or were
with profiles stored in the PulseNet Aotearoa New Zealand Campylobacter
not assigned to any complex. Two of the unassigned isolates
database (16) using an optimization value of 1% and a position tolerance value
of 1.5%. shared at least two alleles with ST-177 and/or ST-682.
Analysis of genotypes. Data for the related wild-bird-associated ST-177 and Further analysis of the 12 ST-45 isolates using PFGE re-
ST-682 complexes were analyzed using the method of Didelot and Falush (8), vealed four SmaI profiles. The largest group (group A) con-
who provided a Bayesian model-based method for inferring clonal relationships tained five isolates, two groups (groups B and C) each con-
of bacteria based on MLST data. The method of these workers was implemented
in the software ClonalFrame (version 1.1). We performed two independent runs
tained three isolates, and one (group D) contained a single
of the Markov chain from different random configurations; each run included isolate. Compared with a database consisting of 318 SmaI
100,000 steps, the first 50,000 steps of which were discarded as burn-in, and the PFGE profile isolates from food, environmental, and human
state was sampled every 100 steps. Initial values and prior values for the param- sources in New Zealand, which included 37 ST-45 isolates, the
eters were all left at the default settings. ClonalFrame was used to assess the
group B profile was indistinguishable from the profiles for two
convergence of the Markov chain by comparing the results of the two indepen-
dent runs using the method of Gelman and Rubin (15). All Gelman-Rubin human ST-45 isolates from clinical cases in Christchurch and
statistics were less than 1.2, indicating that there was satisfactory convergence. Palmerston North (the latter obtained in 2005). The group B
The 500 post-burn-in trees from each of the two runs were concatenated and profile was also indistinguishable from the profiles for three
exported as a Newick file containing 1,000 trees. ST-45 isolates cultured from poultry meat in Wellington and
We then used SplitsTree4 (18) to visualize the 20% threshold consensus
network (17) of the 1,000 post-burn-in trees. The consensus network displayed all
Palmerston North, and the group C profile was indistinguish-
edges (splits) that appeared in more than 20% of the trees. A consensus network able from the profiles for two ST-45 isolates cultured from
can display conflicting splits as boxed areas where sets of parallel edges all poultry meat in Auckland and Hamilton.VOL. 75, 2009 C. JEJUNI FROM WILD BIRDS IN PLAYGROUNDS 781
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FIG. 1. Consensus network for the 1,000 post-burn-in trees from a ClonalFrame analysis of C. jejuni isolates belonging to clonal complexes
ST-177 and ST-682. New Zealand isolates used in this study are indicated by large light blue circles. The threshold was 20%, meaning that all edges
(splits) that appear in more than 200 of the post-burn-in trees are displayed in the network. The areas in boxes represent conflicting splits. B and
green circle, bathing beach; S and dark blue circle, starling (United Kingdom); C and black circle, cat; P and red circle, poultry environment; H
and white circle, human; W and small light blue circle, other wild bird (Europe); WU and small light blue circle, wild bird (United States); U,
unknown.
Of the remaining 10 isolates, 3 were not cut with the SmaI Figure 1 shows two groups of fairly closely related sequence
enzyme and produced a single band (ST-681, ST-2539, and types, one containing ST-177 and one containing ST-682.
ST-583), 5 had unique profiles (ST-137, ST-681, ST-1324, ST- There are also other smaller groups of more divergent strains.
2354, and ST-2357), and the 2 ST-2536 isolates were indistin- Of the sequence types found in New Zealand, ST-2538 and
guishable from each other and their profiles were different ST-2537 fall into the group containing ST-177; ST-681, ST-
from all other profiles. The ST-137 isolate was indistinguish- 2536, and ST-2539 fall into the group containing ST-682; and
able from a single ST-45 poultry isolate in the PulseNet ST-2354 and ST-1324 belong to a more divergent group. This
Aotearoa New Zealand Campylobacter database, and the ST- supports the hypothesis that there were multiple origins of the
2354 isolate was indistinguishable from an ST-991 isolate from campylobacters in the wild-bird population of New Zealand.
a wild duck; all other isolates were unique to this study.
Phylogenetic analysis based on MLST. Our New Zealand DISCUSSION
isolates were compared with other members of wild-bird-asso-
ciated complexes ST-682 and ST-177 from Europe submitted This study demonstrated that fecal material deposited by
to the Campylobacter PubMLST database (http://pubmlst.org wild birds frequenting children’s playgrounds in New Zealand
/campylobacter/). Sequence types belonging to clonal complex may contain C. jejuni, including strains associated with human
ST-45 were not included in this analysis as they are very dis- disease. Approximately one-half of the strains recovered from
tantly related to the ST-177 and ST-682 complexes (there are the fecal material were ST-45 strains, a multilocus sequence
59- and 63-bp differences between ST-45 and ST-177 and ST- type associated with many species of animals, including wild
682, respectively, whereas there are only 8 differences between birds (4–6, 14), and frequently recovered from human cases of
ST-177 and ST-682) and clearly represent a different introduc- campylobacteriosis (http://pubmlst.org/campylobacter/). The
tion into the New Zealand wild-bird population. The consen- SmaI PFGE profile of three of the ST-45 isolates in this study
sus network shown in Fig. 1 contains nine separate areas with was indistinguishable from the profile of isolates recovered
boxes, indicating parts of the tree that are uncertain. However, from human clinical cases, providing evidence that wild-bird
the boxes all indicate local rearrangements, and the overall feces in playgrounds cannot be ruled out as a potential source
structure of the tree is stable. of infection of young children. Further typing using a second782 FRENCH ET AL. APPL. ENVIRON. MICROBIOL.
enzyme, such as KpnI, or flaA short variable region (9) se- ACKNOWLEDGMENTS
quencing could be used to investigate these relationships in We used the Campylobacter Multi Locus Sequence Typing website
more detail. (http://pubmlst.org/campylobacter/) developed by Man-Suen Chan and
Although exposure to wild-bird fecal material is a possible Keith Jolley and located at the University of Oxford; the initial devel-
pathway for human infection, particularly in young children, opment of this site was funded by the Wellcome Trust, and mainte-
nance is funded by DEFRA.
it is unlikely to be a primary contributor to the overall We thank the many contributors to the PubMLST database who
burden of human clinical cases in New Zealand. Food-re- gave us permission to use information for unpublished isolates.
lated exposure, particularly due to handling and consump-
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