1. Risk Analysis DOI: 10.1111/risa.12433
A QMRA for the Transmission of ESBL-Producing
Escherichia coli and Campylobacter from Poultry Farms
to Humans Through Flies
Eric G. Evers,∗
Hetty Blaak, Raditijo A. Hamidjaja, Rob de Jonge, and Franciska M. Schets
The public health significance of transmission of ESBL-producing Escherichia coli and
Campylobacter from poultry farms to humans through flies was investigated using a worst-
case risk model. Human exposure was modeled by the fraction of contaminated flies, the
number of specific bacteria per fly, the number of flies leaving the poultry farm, and the num-
ber of positive poultry houses in the Netherlands. Simplified risk calculations for transmission
through consumption of chicken fillet were used for comparison, in terms of the number of
human exposures, the total human exposure, and, for Campylobacter only, the number of hu-
man cases of illness. Comparing estimates of the worst-case risk of transmission through flies
with estimates of the real risk of chicken fillet consumption, the number of human exposures
to ESBL-producing E. coli was higher for chicken fillet as compared with flies, but the total
level of exposure was higher for flies. For Campylobacter, risk values were nearly consistently
higher for transmission through flies than for chicken fillet consumption. This indicates that
the public health risk of transmission of both ESBL-producing E. coli and Campylobacter
to humans through flies might be of importance. It justifies further modeling of transmission
through flies for which additional data (fly emigration, human exposure) are required. Simi-
lar analyses of other environmental transmission routes from poultry farms are suggested to
precede further investigations into flies.
KEY WORDS: Campylobacter; ESBL-producing Escherichia coli; flies; poultry; risk
1. INTRODUCTION
Pathogenic microorganisms causing human dis-
ease can be transmitted to humans through food,
animals, the environment, and other humans. It is
important to quantify the attribution of this transmis-
sion between and within these sources to support a
government considering interventions to reduce pub-
lic health risk. Havelaar et al.(1)
described that for 14
pathogens that can be transmitted by food, 38% of all
Centre for Zoonoses and Environmental Microbiology, National
Institute for Public Health and the Environment, Bilthoven, The
Netherlands.
∗Address correspondence to Eric G. Evers, cZ&O, RIVM, P.O.
Box 1, 3720 BA Bilthoven, The Netherlands; tel: +31 30 2744149;
fax: +31 30 2744434; eric.evers@rivm.nl.
Dutch human disease cases is caused by foodborne
transmission.
Within foodborne transmission, chicken con-
sumption is a major contributor as it is esti-
mated to cause 20–40% of all campylobacterio-
sis cases.(2)
The relevance of transmission from
poultry to humans through the food chain was
also suggested by Leverstein-van Hall et al.(3)
in
relation to antimicrobial resistance. They demon-
strated among a set of representative clinical
extended-spectrum β-lactamase (ESBL)-producing
Escherichia coli isolates from the Netherlands,
variants that were indistinguishable from isolates
from Dutch poultry and chicken meat, with re-
spect to ESBL-gene, plasmid type, and strain
genotype.
1 0272-4332/15/0100-0001$22.00/1 C 2015 Society for Risk Analysis
2. 2 Evers et al.
Besides foodborne transmission, transmission
through the environment might also be relevant
for spread of poultry-associated zoonoses. Friesema
et al.(4)
found indirect support for significant envi-
ronmental transmission of Campylobacter to humans
when analyzing an outbreak of avian influenza in
poultry, which resulted in extensive culling, espe-
cially of laying hens, and closing of slaughterhouses.
These interventions led to a large reduction in human
campylobacteriosis cases that could not be explained
by the simultaneously occurring minor reduction in
chicken meat consumption.
A number of studies suggest that environmen-
tal transmission of pathogens to humans through
flies may be relevant. Ekdahl et al.(5)
listed six ar-
guments supporting the hypothesis of transmission
of Campylobacter through flies to humans: the low
infective dose, the ability of flies to function as a
vector, the ubiquitous presence in the environment,
the seasonality of campylobacteriosis, the age pattern
for campylobacteriosis in Western travelers to the
tropics, and the dominance of solitary human cases.
Based on this hypothesis, Nichols(6)
showed that the
seasonal character of campylobacteriosis was related
to the larval development time of the house fly Musca
domestica. Other studies focused on transmission of
pathogens to and between poultry farms as a result of
fly movements. Bahrndorff et al.(7)
found that placing
fly screens at broiler houses resulted in a reduction of
the percentage Campylobacter positive flocks from
41% to 10%. Hansson et al.,(8)
however, could not
find a relationship between the Campylobacter inci-
dence in broiler flocks and the presence of Campy-
lobacter in the environment (ground, insects, water,
feed, and ventilations shafts). Still, transmission of
Campylobacter from chicken to flies and vice versa
was indeed proven experimentally by Shane et al.(9)
The aim of this study was to investigate quan-
titatively whether transmission of ESBL-producing
E. coli and/or Campylobacter from poultry farms
through flies to humans could be a relevant transmis-
sion route in terms of public health risk.
2. MATERIALS AND METHODS
2.1. General
We used a quantitative microbial risk assessment
(QMRA) approach based on field and literature data
to estimate the public health risk of ESBL-producing
E. coli and Campylobacter on flies in the poultry
farm environment. The fly model considers events
up to and including emigration rate of the flies when
leaving the farm followed by a worst-case approach,
assuming all bacteria (ESBL-producing E. coli or
Campylobacter) on/in the flies are transmitted to hu-
mans in the vicinity of the farm. The relevance of
transmission through flies was assessed through com-
parison with QMRA estimates of exposure to and
risk of transmission through consumption of chicken
fillet, based on the idea that if worst-case fly risk is
lower than real chicken fillet risk, then fly risk is neg-
ligible in terms of public health. For ESBL-producing
E. coli only exposure was considered as there is as yet
no theoretical framework available to estimate the
type and number of human adverse health outcomes.
We chose chicken fillet for comparison as it is con-
sidered to be a food product with significant public
health risk(2,3)
for which sufficient data are available
and to a lesser extent as it also concerns chickens.
We compared the QMRA for flies with a QMRA for
chicken fillet and not with epidemiological estimates
in order to have a consistent methodology that allows
for a comparison of ESBL-producing E. coli expo-
sure estimates through both transmission routes. Re-
sulting much lower worst-case QMRA estimates for
flies compared to the QMRA estimates for chicken
fillet would make transmission through flies a neg-
ligible route; otherwise, further research would be
deemed sensible.
2.2. The Fly Model
In the model, we describe a worst-case sce-
nario for flies transmitting ESBL-producing E. coli
or Campylobacter from poultry farms (laying hens,
broilers) to humans who are present in the vicinity
of the farms (close enough to be reached by flies).
A conceptual model of the processes playing a role
in this transmission is shown in Fig. 1. In short, flies
enter the poultry farm from outside the farm area
(immigration) or develop into adults at the poultry
farm, become contaminated with ESBL-producing
E. coli and/or Campylobacter by ingestion of or con-
tact with contaminated chicken feces, leave the poul-
try farm (emigration), reach humans in the vicinity,
and transmit the bacteria to humans. This transmis-
sion can occur by mechanical dislodgement from the
flies’ exoskeleton, fecal deposition, and regurgitation
of food.(10)
At a poultry farm, one or more poultry
houses may be present, and the flies can become con-
taminated in the vicinity of the poultry house, for
example, through foraging at dung heaps or contact
3. QMRA for Transmission from Poultry Farms Through Flies 3
Fly population on farm
+: Reproduction
‐ : Death (e.g., old age, predation)
Contamination of flies with E/C
(given a contaminated farm)
Fraction of contaminated flies
No. of cfu per contaminated fly
Immigration
Emigration
Inactivation E/C
Death (old age, predation)
Fly lands on food, part of E/C
transmitted to food, food ingested
(For Campylobacter)
Dose response to illness
Cases of ilness
Farm
Environment
Human exposure
Other transmission routes
Fly is ingested
Fly lands on hand, part of E/C
transmitted to hand, touch mouth
with hand
Probability of
transmission routes
Human risk
•
•
•
•
•
•
•
•
Fig. 1. Conceptual model of transmission of bacteria from poultry farms through flies to humans. The model only considers processes up to
and including emigration, and uses a worst-case approach for the remainder of the transmission routes, assuming all bacteria in emigrating
flies to be ingested by humans. E/C = ESBL-producing E. coli / Campylobacter.
with stored manure. Flies can also enter the poultry
house itself and become contaminated, but this will
probably be less relevant for transmission to humans,
as only a small part of these flies will leave the poul-
try house. There will be a difference between laying
hen and broiler houses, these being different types of
buildings, but this is not taken into account.
We only model the process up to and including
emigration rate of the flies when leaving the farm,
followed by a worst-case approach for simplicity and
due to lack of data on emigration. We use the follow-
ing assumptions:
(i) Flies (whether or not originating from outside
the farm) can become contaminated with bac-
teria (ESBL-producing E. coli or Campylobac-
ter) on contaminated farms.
(ii) Flies may leave poultry farms.
(iii) Bacteria in/on flies are not inactivated during
emigration from poultry farms to humans.
(iv) All poultry houses at a farm are contaminated
with ESBL-producing E. coli.
(v) All laying hen houses at a farm are contami-
nated with Campylobacter.
(vi) Every separate dose of bacteria on/in a con-
taminated fly that leaves the poultry farm is in-
gested by humans.
The model output is the frequency of hu-
man exposure, the total human exposure, and (for
Campylobacter) the number of human cases of
illness.
For the model description we first consider a
farm with one poultry house, which is contaminated
with bacteria. The number of flies leaving the farm
per day during the fly season is termed fday. The num-
ber of flies leaving the farm in a year fyear equals:
fyear = fdaytfly,
where tfly is the time length of the fly season in days.
The fly season is defined by a much higher number
of flies on the farm compared to the rest of the year.
The number of contaminated flies leaving the farm in
a year f cont
year (= the number of exposures) equals:
f cont
year = fyear pfly,
where pfly is the fraction of contaminated flies given
a farm with a contaminated poultry house. The to-
tal number of bacteria (ESBL-producing E. coli or
Campylobacter) that is transmitted to humans in the
vicinity (within reach of flies) of the farm in a year
dyear (= the total exposure) equals:
dyear = f cont
year dfly,
where dfly is the number of bacteria in/on a contami-
nated fly.
The number of human cases of illness (campy-
lobacteriosis) in the vicinity of the farm in a year cill
equals:
cill = pill f cont
year ,
4. 4 Evers et al.
where pill is the probability of illness after ingesting
the dose of bacteria in/on a contaminated fly, which
equals:
pill = pill|inf pinf ,
where pill|inf is the probability of illness given in-
fection and pinf is the probability of infection after
ingesting the mean dose of bacteria in/on a contam-
inated fly, which is described by the Beta Poisson
dose-response model:(11)
pinf = 1 − 1 +
dfly
β
−α
,
with α and β parameters.
All model outputs above are outputs per poultry
house, as more than one poultry house may be lo-
cated on a farm. Note that this does not mean that
flies originating from inside the poultry houses are
the main risk. On the contrary, we think that the
main contribution to transmission will be due to flies
that become contaminated on the farm, but in the
environment of the poultry house. To obtain results
for the Netherlands as a whole, we multiplied with
the number of contaminated poultry houses in the
Netherlands. So the number of contaminated flies
leaving contaminated poultry farms in a year in the
Netherlands Fcont
year , the total number of bacteria that
is transmitted to humans in the vicinity of a contami-
nated poultry farm in a year in the Netherlands Dyear,
and the number of human cases of illness in the vicin-
ity of a contaminated poultry farm in a year in the
Netherlands Cill equal:
Fcont
year = f cont
year Nph pph,
Dyear = dyear Nph pph,
Cill = cill Nph pph,
where Nph is the number of poultry houses in the
Netherlands and pph is the fraction of contaminated
poultry houses in the Netherlands.
2.3. The sQMRA Chicken Fillet Model
The swift quantitative microbiological risk as-
sessment (sQMRA) model is a simplified QMRA
approach using a food chain model with measure-
ments of a food product in retail as the starting
point and human cases as the end point. The first de-
terministic version(12)
focused on heating and cross-
contamination during food preparation by the con-
sumer. Chardon and Evers(13)
presented a second
(extended) probabilistic version, which in addition
includes variability of food treatment by the con-
sumer, growth or inactivation during food storage at
the consumers’ home, and the D/z-heating model (14)
for food preparation. In that paper, an example
calculation for Campylobacter on chicken fillet was
given, the results of which are used here. For ESBL-
producing E. coli on chicken fillet, analogous calcula-
tions are done with the bacterium-specific parameter
values adjusted.
2.4. The Poultry Farm Field Study
2.4.1. Measurements
Data for pfly (the fraction of contaminated flies
given a farm with a contaminated poultry house)
and dfly (the number of bacteria in/on a contami-
nated fly) originate from a study on the prevalence
and number of ESBL-producing E.coli and Campy-
lobacter on flies on Dutch poultry farms.(15,16)
Dur-
ing 2011 and 2012, three broiler farms and five laying
hen farms were visited and sampled. At one of the
broiler farms, flies were collected during four differ-
ent visits (two times during the presence of broilers
and two times during/after cleaning in the absence
of broilers), at the remainder of the farms, flies
were collected during one visit during which chickens
were present. In total, 326 flies (of which 212 Musca
domestica) were caught at the farms, in or near
poultry houses (e.g., manure storage, canteen, egg
sorting area). Flies were collected, transported, and
processed as described by Blaak et al.(15)
Flies were analyzed in pools, each consisting of
one to eight flies of the same species and collected at
the same location. All 326 flies were analyzed for the
presence of ESBL-producing E. coli (73 pools), 297
(of which 202 Musca domestica) were analyzed for
Campylobacter (65 pools). Flies were homogenized
in PBS/0.5% Tween20. For the isolation of ESBL-
producing E. coli, fly homogenates were plated on
ChromID ESBL medium (Biomerieux, Boxtel, The
Netherlands) and 10 mL of the fly homogenates was
additionally enriched in BPW/1 μg/mL cefotaxime
followed by plating on ChromID ESBL medium. In-
cubations were performed for 4 to 5 hours at 36 ±
2 °C, followed by 21 ± 3 hours at 44 ± 0.5 °C. Sus-
pected ESBL-producing isolates were confirmed as
ESBL-producers using disk-diffusion and sequencing
of ESBL-genes.(15)
For the isolation of Campylobac-
ter 10 mL portions of fly homogenates were enriched
in Preston broth, followed by plating on CCDA agar
(Oxoid, Landsmeer, The Netherlands). Incubations
5. QMRA for Transmission from Poultry Farms Through Flies 5
of enrichments in Preston broth as well as CCDA
agar plates were performed microaerobically using
CampyGen sachets (Oxoid B.V., Landsmeer, The
Netherlands), for 44 ± 4 hours at 41.5 ± 1 °C.
On fly collection days, manure and/or wastewa-
ter from the farm was also collected to test for ESBL-
producing E. coli and Campylobacter.
2.4.2. Calculations
We estimate the value of pfly (the fraction of
contaminated flies given a farm with a contaminated
poultry house) through maximizing the likelihood
equation L, where, assuming that pfly is a constant
for positive farms:
L =
i
ni
ki
pki
fly(1 − pfly)ni −ki
,
which is simply the product of binomial distributions
per pooled sample, with ni and ki the total number
and the number of positive flies in pool i, respec-
tively. This can be simplified to:
L = (1 − pfly)n0
i
(1 − (1 − pfly)ni,pos
)
or
ln L = n0 ln(1 − pfly) +
i
ln(1 − (1 − pfly)ni,pos
),
where the part of the equation left of the +sign repre-
sents negative pools with n0 the total number of flies
in negative pools and the part of the equation right
of the +sign represents the positive pools with ni,pos
the number of flies in positive pool i.
The number of bacteria (ESBL-producing E. coli
or Campylobacter) in a positive fly in pool i, dfly,i, is
calculated with:
dfly,i =
ni,pos
j=1 Bin j|ni,pos, pfly
dpool,i
j
1 − Bin(0|ni,pos, pfly)
,
where dpool,i is the total number of bacteria in pool
i. The formula calculates the average number of bac-
teria in a positive fly, weighted with the probability
of j positive flies in the pool. The number of bacteria
in a positive fly, dfly, is then calculated by taking the
average of the dfly,i values.
All calculations were done with Microsoft Excel,
using the Solver function to estimate pfly.
2.5. Parameter Values
For all fly model parameter values other than
ESBL-producing E. coli pfly and dfly, and for the
chicken fillet model parameter values, data or ad-
ditional data were mainly obtained from literature,
but also from unpublished own research, and through
personal communication. The estimated parameter
values are given in Tables I–V. The data used to ob-
tain these values are described in Sections 3.1–3.4.
3. RESULTS
3.1. Fly Migration Behavior Parameters
fday (the number of flies leaving the farm per day)
The influx of flies (Diptera: Brachycera) into
broiler houses was estimated by Hald et al.(17)
at 6,524
± 638 (standard error) flies per broiler rotation, or
186 flies per day, setting 35 days per rotation. Hald
et al.(18)
found 917 flies per day during the last week
of rotation, which was explained by the increased vol-
ume (m3
/hours) of ventilation air inflow. We used
the value of Hald et al.(17)
as an estimate of the flux
of flies towards the poultry house and its possibly
contaminated vicinity (e.g., dung and manure heaps),
the flies originating from the wider farm terrain or
abroad (e.g., other farms). This value was used as the
basis to estimate fday, the number of flies per day leav-
ing the farm and possibly contaminating humans.
For this, release-recapture experiments of flies
on farms were used as these give an estimate of the
fraction of released flies that remain on and that
leave the farm. Lysyk and Axtell(19)
released marked
house flies in the poultry house on two livestock farm
locations containing a dairy, poultry houses, build-
ings, pastures, and ungrazed fields. Of the flies re-
captured after five days, a fraction of 0.17 came from
field and pasture. Recaptured fly numbers were low
farther than 50 m away from the release point, so the
value of 0.17 constitutes a maximum estimate, also as
flies that are eaten will not be recaptured.
Using the data above, the number of flies leaving
the farm per day fday was estimated at 0.17 × 186 =
32.
tfly (duration of the fly season in days)
We will work from the starting point of a
season-dependent fly population size, although on
a cattle/pig farm in Denmark, Kristiansen and
Skovmand(20)
found a relation of fly population size
with manure presence rather than with the month
of the year. Hald et al.(18)
stated that the fly season
6. 6 Evers et al.
Table I. Parameter Values of the Fly Model
Parameter ESBL-Producing E. coli Campylobacter
Symbol Definition Broiler Laying Hen Broiler Laying Hen
fday Number of flies leaving the farm per day 32 32 32 32
tfly Duration of the fly season in days 91 91 91 91
pfly Fraction of contaminated flies given a
farm with a contaminated poultry
house
0.048 0.031 0.137 0.137
dfly Number of bacteria in/on a contaminated
fly
1.96E4 1.28E3 2.5E2 2.5E2
pill|inf
The probability of illness given infection – – 0.33 0.33
α Parameter of the Beta Poisson
dose-response model
– – 0.145 0.145
β Parameter of the Beta Poisson
dose-response model
– – 7.589 7.589
Nph Number of poultry houses in the
Netherlands
2212 1974 2212 1974
pph Fraction of contaminated poultry houses
in the Netherlands
1 1 0.45 1
around broiler houses stretches from April to Octo-
ber, with a peak in abundance and activity in July
and August. Meerburg et al.(10)
stated that house
flies (Musca domestica) can be found near pig farms
from spring to October, with 10–12 generations of
15–25 days. Skovgard and Nachman(21)
found a large
population of stable flies (Stomoxys calcitrans) on a
dairy farm from June/July to September. So flies will
be present for about seven months per year, but in
higher numbers for some three months (= 91 days),
which we will use as a value for tfly.
3.2. Contamination of Flies and Human
Dose-Response Parameters
3.2.1. Poultry Farm Field Study
All farms were (manure/wastewater)-positive for
ESBL-producing E. coli at fly sampling days. The
data on absence/presence and numbers of ESBL-
producing E.coli in flies are shown in Tables II and
III together with the estimated pfly (the fraction of
contaminated flies given a farm with a contaminated
poultry house) and dfly (the number of bacteria in/on
a contaminated fly).
The higher mean number of ESBL-producing
E. coli in flies from broiler farms compared to lay-
ing hen farms is in agreement with measurements in
fresh chicken feces sampled at the same time as the
flies. Geometric mean concentrations were 2.3 × 104
CFU/g in broiler faeces (39 samples, three farms) and
5.0 × 102
CFU/g in laying hen feces (27 samples, five
farms).(16)
All laying hen farms and two out of three broiler
farms were Campylobacter-positive; at the farm that
was sampled multiple times, Campylobacter was de-
tected at one of the four visits. For 13 of 22 fly pools
from broiler farms, the farms were Campylobacter-
negative at time of sampling. All flies were negative
for Campylobacter (Table IV).
3.2.2. Literature Data
In the literature we found no data on ESBL-
producing E.coli in flies, but in a number of stud-
ies Campylobacter in or on flies was investigated,
showing a very large range of the percentage of
Campylobacter positive flies pfly (Table IV). We did
not include fly prevalence data from other farms
than laying hens and broilers. In the investigation
of Rosef and Kapperud,(22)
the pathogen was identi-
fied as Campylobacter fetus subsp. jejuni from house
flies, which is interpreted as Campylobacter spp. (per-
sonal communication, Jacobs-Reitsma, RIVM, The
Netherlands). Berndtson et al.(23)
did not specify the
exact pool sizes; pooled samples contained one to six
flies. The results were analyzed with the same maxi-
mum likelihood approach as described in Section 2.4,
taking the mean of scenarios of pools of size one
to six to obtain the estimates in Table IV. Hansson
et al.(8)
did investigate the broiler farms for Campy-
lobacter spp. but the accumulated data do not allow
7. QMRA for Transmission from Poultry Farms Through Flies 7
Table II. Data on Number of Collected Flies, Their ESBL-Producing E. coli Status and the Estimated pfly
Variable Description Broiler Farm Laying Hen Farm
Total no. of pools 29 44
No. of negative pools 24 38
No. of positive pools 5 6
Total no. of flies 117 209
No. of negative flies 88 178
Pool size of positive pools 3, 8, 8, 2, 8 8, 8, 8, 2, 4, 1
estimated pfly 0.048 0.031
Table III. Data on Detected Numbers of ESBL-Producing E. coli in Fly Pools, and Estimated dfly,i and dfly
Poultry Pool Size ESBL-Producing Estimated Estimated
Farm (No. of E. coli Number in Pool dfly,i dfly
Type Flies) (CFU/Pool) (CFU/Positive Fly) (CFU/Positive Fly)
Broiler 3 2.54E+04 2.48E+04 1.96E+04
8 4.95E+02 4.53E+02
8 1.65E+02 1.51E+02
2 1.98E+03 1.96E+03
8 7.69E+04 7.04E+04
Laying hen 8 1.16E+03 1.09E+03 1.28E+03
8 4.95E+02 4.68E+02
8 8.41E+01 7.95E+01
2 4.13E+03 4.09E+03
4 8.25E+02 8.06E+02
1 1.16E+03 1.16E+03
to relate the Campylobacter status of the flies to
the Campylobacter status of the farms. However, the
farms could be distinguished into often, intermedi-
ate, or rarely positive in the past three years and we
excluded the last category for the calculation of the
mean Campylobacter prevalence of flies. Again, for
the individual fly prevalence estimates we took the
mean of maximum likelihood estimates for pools of
size 1–25, as pool size was not exactly specified.
Few or none of the data in Table IV would be
useful to estimate pfly, the fraction of contaminated
flies given a farm with a contaminated poultry house,
when strict criteria would be applied. These criteria
are: (i) the poultry farms are to be Campylobacter
positive and (ii) no other Campylobacter positive an-
imals are to be present in the vicinity (as these could
also contaminate the flies). We decided to use the
majority of the data in Table IV, accepting the short-
comings, in order to obtain a rough estimate of pfly,
excluding Campylobacter- negative farms where pos-
sible, and the “farm near pigs” from Hald et al.(17)
as the pigs were probably Campylobacter- positive.
Also, we excluded the PCR data (as only viable
campylobacters are relevant) and did not distinguish
between Campylobacter species. The considerations
above resulted in an estimated mean value of 0.137
for pfly.
There are no data on numbers of Campylobac-
ter on or in flies (dfly). Therefore, we estimated these
numbers from data on food consumption rate and
activity time span of flies, and the concentration of
Campylobacter in chicken feces. It is thus assumed
that the amount of Campylobacter on the surface of
a fly is of less importance than the campylobacters in
its inside, based on Shane et al.(9)
They exposed flies
(Musca domestica) to a Campylobacter solution and
isolated Campylobacter from the feet and ventral sur-
face of 14% of the flies, and from the viscera of 81%
of the flies.
Kobayashi et al.(24)
measured an ingested
amount of trypticase soy broth by houseflies (Musca
domestica vicina) of 3.35 μL ࣈ 3.35 mg after 30 min.
Shepard et al.(25)
measured a maximum of 6 hours of
activity of flies (Musca domestica) in 8 hours. This
equals 9 hours of activity for a daylight length of
12 hours. Considering one day and assuming all ac-
tivity is feeding, the ingested volume is 9 × 2 ×
3.35 = 60 mg. Hutchison et al.(26)
found the mean
9. QMRA for Transmission from Poultry Farms Through Flies 9
Campylobacter concentration in positive fresh poul-
try feces in the United Kingdom to be 4.2E3 CFU/g.
So then dfly, the number of Campylobacter in a con-
taminated fly, can be estimated to be 60E−3 g ×
4.2E3 CFU/g = 2.5E2 CFU. This is a maximum es-
timate in the sense that this number will in reality be
lower due to regurgitation, defecation, and inactiva-
tion in the alimentary tract.(27)
The effect of ingested campylobacters on hu-
mans is described in terms of the probability of in-
fection pinf and illness pill. The equations are given in
Section 2.2. The parameter values used are α = 0.145,
β = 7.589,(11)
and pill|inf = 0.33,(28)
based on human
volunteer studies.
3.3. Poultry Farm Parameters
The number of active broiler and laying hen
farms in the Netherlands in 2013 was 774 and 896, re-
spectively. The average number of broiler and laying
hen houses per farm was 2.86 and 2.20, respectively.
So the number of broiler and laying hen houses in the
Netherlands Nph was estimated at 2,212 and 1,974,
respectively (personal communication, Erik Bout,
Dutch Product Boards for livestock, meat, and eggs).
Dierikx et al.(29)
measured ESBL-producing
E.coli in 26 broiler farms in the Netherlands in the
period March–June 2009. Based on 25–41 cloaca
swabs per farm, all farms were found positive for
ESBL-producing E. coli. Huijbers et al.(30)
found all
50 broiler farms investigated in the Netherlands in
July 2010–April 2011 positive for ESBL-producing E.
coli. This was based on cloaca swabs from 20 broilers
taken from all houses from a farm, with a minimum
of one positive pool sample of two swabs as a cri-
terion. In our field study we found ESBL-producing
E. coli in feces in all investigated broiler and laying
hen farms. Based on all the above observations, pph
(the fraction of contaminated poultry houses in the
Netherlands) was set to 1 for broiler and laying hen
farms.
Jore et al.(31)
gave monthly surveillance data for
Campylobacter in Dutch broiler flocks for the period
2001–2007. The highest mean prevalence for a three-
month period was reported for June–August or July–
September and equaled 0.45. This value was chosen
for pph. It agrees with the six of 12 (50%) positive
sampling events at three broiler farms in 2011–2012
in the poultry house field study.
The Campylobacter prevalence for laying hen
flocks is unknown. Given that laying hens get rel-
atively old (about 80 weeks, whereas broilers are
culled after six weeks), the assumption that these an-
imals are Campylobacter positive for the main part
of their life seems realistic (personal communication,
Jacobs-Reitsma, RIVM, The Netherlands). This is in
agreement with our field study where we found 10 of
10 (100%) positive sampling events at five laying hen
farms in 2011–2012. Therefore, pph is set to 1.
3.4. Chicken Fillet Parameters
The ESBL-producing E. coli data used for the
sQMRA calculations are given in Table V. Data
for percentage contamination and concentration
of ESBL-producing E. coli on conventional retail
chicken fillets were obtained from two Dutch stud-
ies. A lognormal distribution was fitted to the con-
centration data of each study. For 60 chicken fillets
sampled in 2010(32)
this resulted in 100% contami-
nated chicken fillets with mean and standard devia-
tion for the ESBL-producing E. coli concentration of
11.4 and 48.0 CFU/g, respectively. Of 140 chicken fil-
lets sampled in 2011, 99.3% were contaminated with
ESBL-producing E. coli and mean and standard de-
viation for the ESBL-producing E. coli concentration
were 5.8 and 31.9 CFU/g, respectively (unpublished
data, Dutch Consumers Association/RIVM). For the
sQMRA calculation we used the average values from
both studies for the three parameters: 99.7% contam-
inated chicken fillets and a mean and standard devi-
ation for the concentration of 8.57 and 39.92 CFU/g,
respectively.
3.5. Comparison of the Risk of Transmission
Through Flies and Chicken Fillet
The model outputs in terms of human expo-
sure for ESBL-producing E. coli and human expo-
sure and illness for Campylobacter through contact
with flies (broiler, laying hen) and consumption of
chicken fillet are shown in Table VI. Note that the
number of exposures is the number of contaminated
flies originating from a poultry farm, or the number
of consumed contaminated chicken fillets, in a year.
The total exposure is the number of ingested ESBL-
producing E. coli or Campylobacter in a year. Note
also that the fly estimates are worst-case estimates,
which is illustrated by the Campylobacter results at
poultry-house level: we estimate the very high value
of 52 campylobacteriosis cases per year per positive
broiler or laying hen house, 52 cases being caused by
394 exposures.
10. 10 Evers et al.
Table V. ESBL-Producing E. coli Parameter Values Used for the sQMRA Calculations
Parameter Subgroup Parameter Description Parameter Value Explanation
Contamination level at retail Percentage of contaminated
chicken fillets
99.7 % For explanation see text.
Mean concentration 8.57 CFU/g
Standard deviation of
concentration
39.92 CFU/g
Growth and inactivation
during storagea
Minimum generation time 0.30 hours Value for ground mutton from Table 1b
“Temperature and Growth.”
Optimum growth temperature 37.5 °C From Table D “Limits for growth.”
Minimum growth temperature 7.5 °C From Table D “Limits for growth.”
Probability of survival per
CFU and day at room
temperature
1 d−1 Assumption based on the fact that Table
1a “Temperature and survival” gives
no data at room temperature.
Probability of survival per
CFU and day in the
refrigerator
0.896 d−1 Based on the reported reduction of about
a factor 10 in about three weeks in the
section “Growth and survival
characteristics.”
Probability of survival per
CFU in the freezer
0.1 Based on the reported reduction of a
factor of 10 after 38 weeks at −25.5 °C
for nonpathogenic E. coli in the section
“Growth and survival characteristics.”
Inactivation due to heating
during food preparation
D value at the reference
temperature Dref
0.21 min Using the values for E. coli from van
Asselt and Zwietering.(14)
z value 10.6 °C
Reference temperature Tref 70 °C
aData were obtained from Chapter 7 “Intestinally pathogenic Escherichia coli” from ICMSF.(37)
Table VI. Comparison of Exposure and Illness Between Transmission Through Flies (Worst-Case Approach) and Chicken Fillet
No. of Total Exposure No. of Cases
Transmission Exposures (No. of Bacteria) (cill, Cill)
Bacterium Route Level Type ( f cont
year ,Fcont
year ) (dyear, Dyear)
ESBL-producing
E. coli
Fly Positive poultry
housea
Broiler 1.4E+02 2.7E+06
Laying hen 8.9E+01 1.1E+05
The Netherlands Broiler 3.1E+05 6.0E+09
Laying hen 1.8E+05 2.3E+08
Chicken fillet The Netherlands – 4.1E+06 1.5E+08
Campylobacter Fly Positive poultry
housea
Broiler 3.9E+02 9.9E+04 5.2E+01
Laying hen 3.9E+02 9.9E+04 5.2E+01
The Netherlands Broiler 3.9E+05 9.8E+07 5.2E+04
Laying hen 7.8E+05 1.9E+08 1.0E+05
Chicken fillet The Netherlands – 4.6E+05 1.8E+07 1.1E+04
aIncluding the vicinity of the poultry house.
Focusing on public health risk, thus on the level
of the whole of the Netherlands, for ESBL-producing
E. coli the number of exposures is higher for chicken
fillet than for flies, but the total exposure is higher
for flies than for chicken fillet (Table VI). For broiler
flies, total exposure is a factor 40 higher than for
chicken fillet; for laying hen flies a factor 1.5. The
higher values for broiler flies compared to laying hen
flies are mainly caused by the higher pfly (the fraction
of contaminated flies given a farm with a contami-
nated poultry house) and dfly (the number of bacteria
in/on a contaminated fly).
For Campylobacter, the number of exposures
through chicken fillet is in between that through flies
11. QMRA for Transmission from Poultry Farms Through Flies 11
from broilers and laying hens (Table VI). The total
exposure and the number of cases is higher for flies
than for chicken fillet, with a factor of about 5 for
broiler flies and about 10 for laying hen flies. The
higher values for laying hen flies compared to broiler
flies are caused by the higher laying hen value of pph
(the fraction of contaminated poultry houses in the
Netherlands).
4. DISCUSSION
Our results, which are based on the currently
available knowledge, imply that transmission of both
ESBL-producing E. coli and Campylobacter through
flies to humans cannot be considered a negligible
public health risk and further research is sensible.
The basis for this conclusion is a comparison of a
worst-case risk assessment for transmission of bac-
teria through flies with a simplified risk assessment
for transmission of bacteria through consumption
of chicken fillet. A worst-case approach implies of
course that the real value can be any value lower than
or equal to the estimated value. An obvious next step
would be the replacement of the worst-case estimates
for transmission through flies with realistic estimates,
while retaining the relative risk approach by compar-
ing with the risk of chicken fillet consumption. This
would comprise of an improvement of the emigration
value fday and extension of the model to incorporate
processes during and after emigration.
An alternative approach to estimate the emigra-
tion flux of flies would be to use the results of release-
recapture studies. In our opinion, these studies are,
however, suitable to estimate relative numbers rather
than absolute fluxes of flies. This relates to the com-
plexity of the dynamics of the fly population on a
farm, which includes population size, birth rate, pre-
dation, death rate, immigration, emigration, and ei-
ther or not a steady state situation together with the
unknown fraction of nonrecaptured flies. Therefore,
we preferred to use the number of 186 flies per day(17)
as a basis with the important advantage of being a
real measured number. We are aware of course this
is a poultry house influx number, but we see it as a
number that gives an idea of the size of immigration
and emigration fluxes that is usable for a worst-case
QMRA approach. Next, we assumed that this num-
ber of 186 flies can be seen as analogous to released
flies in a release-recapture study. We used the result
of the Lysyk and Axtell study,(19)
which can be in-
terpreted as that a maximum of 17% of the flies will
leave the farm.
Processes during and after emigration include
(Fig. 1): inactivation of ESBL-producing E. coli and
Campylobacter on/in flies during their flight to hu-
mans; death of the flies before reaching humans; the
probability of actually reaching humans; the proba-
bility of each possible transmission route to occur;
and properties of specific transmission routes (e.g.,
fraction of bacteria transmitted from flies to human
food). These are all aspects with no or scarce data
availability, which was an important reason to follow
the worst-case approach applied here.
Skovgard et al.(27)
did study the inactivation
of Campylobacter in artificially inoculated flies, but
their results do not allow for a quantitative estimate
of Campylobacter inactivation rate as only absence or
presence was measured, which in addition is related
to the detection limit. An example result is that at low
and high dose and 15 °C 1% of the flies are still car-
riers ca. 48 hours and 60–70 hours after inoculation,
respectively.
The probability of flies actually reaching humans
will be related to human population density as a func-
tion of the distance from the poultry farms and the
fly range of the flies. Data on fly range of flies are,
however, limited. Based on laboratory experiments
of Shepard et al.,(25)
the flight range per day for five-
day-old flies can be estimated at 5.4 km. Recapture
field experiments at a poultry farm by Nazni et al.(33)
showed that a flight distance of 4 km or more seldom
occurs.
The uncertainty of pfly, the fraction of
Campylobacter- positive flies given a contami-
nated poultry house, obtained from the poultry
house field study and literature data, is large (Table
IV). It was noted that the percentage of positive
flies decreased with the publication year of the
study, being higher in older studies and lowest in
more recent studies. The Campylobacter detection
method was more or less similar in all studies;
however, high percentages of positive flies were
found in the older studies where laboratory analysis
started within four or six hours after fly capture,(22,23)
this period being 24 hours or longer in the more
recent studies. Live transportation of the flies to
the laboratory, as practiced and stated to be im-
portant for reliable Campylobacter detection,(17,18)
does not give high pfly values in combination with
these longer capture-analysis time periods. Possi-
bly, the Campylobacter die-off on/in flies is rapid,
which is supported by the much higher percentage
of positive flies detected by PCR as compared to
conventional culture.(18)
In our opinion, the data did
12. 12 Evers et al.
not sufficiently support the use of the higher values
only(22,23)
(which besides are unlikely to be false pos-
itives). Therefore, we chose to use the mean value
of the percentages Campylobacter obtained in the
various studies. Alternatively, this can be regarded
as implicitly including Campylobacter inactivation
during the flight of the flies from the poultry farm to
humans.
A possibly relevant extension of the model could
be to distinguish between free-range poultry farms
and farms where chickens stay inside. When chickens
are outside, their (fresh) feces are easier accessible
for flies. In addition, Campylobacter flock prevalence
in free-range chickens and chickens at conventional
farms may differ. Hoogenboom et al.(34)
reported
Campylobacter presence in feces at all nine sam-
pled organic free-range broiler farms (100%) in 2005,
whereas the Campylobacter prevalence was 22% in
289 conventional broiler flocks in 2001 to 2002.(35)
When transmission through flies would prove to
be of significant importance, the development of in-
tervention strategies becomes relevant. The use of
fly screens, which was shown to reduce Campylobac-
ter broiler flock prevalence from 41% to 10%,(7)
is a possibility (although not for free-range poultry
farms). This would extend the functionality of these
screens from prevention of transmission between
poultry farms to include prevention of transmission
from poultry farms to humans. Another possibility
would be to reduce the size of the fly popu-
lation on poultry farms by preventive measures,
such as rapid removal of waste and manure, stor-
ing waste in well closable containers and keeping
manure covered, and cleaning the poultry houses
regularly.
The study of Friesema et al.(4)
suggests a signif-
icant transmission of Campylobacter from poultry
farms to humans through the environment. Apart
from flies, air, soil, and water are also relevant for
environmental transmission of bacteria, with water
and air presumably being the most relevant. From
poultry farms, bacteria can enter the surface water
surrounding the farm by runoff to ditches. Humans
can be exposed to contaminated surface water
during swimming activities, or through consumption
of irrigated agricultural crops. An estimation of the
relative importance of the different environmental
transmission routes can be obtained by performing
worst-case or simplified QMRA calculations (e.g.,
Evers et al.(36)
). A subsequent extensive QMRA of
the apparent most important environmental trans-
mission route then is to result in recommendations
for risk management on interventions to reduce
public health risk of ESBL-producing E. coli and
Campylobacter.
ACKNOWLEDGMENTS
The authors thank Lianne Kerkhof - de Heer
and Angela van Hoek for their assistance in the field
and laboratory work, and Wilma Jacobs-Reitsma for
helpful discussions. This study was conducted on be-
half of the Netherlands Food and Consumer Product
Safety Authority.
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