A high relative frequency of a variable being included in the
various models, and a consistent association with flock colonization
across models (Table 4),
may help indicate the true causal role of that factor, and hence the
potential for producers to decrease risk on the farm by applying an
appropriate intervention directed at that factor. Median flock size,
followed by farm water source and the presence of other domestic
livestock on the farm, were the factors that were included in most or
all of the models. Spreading manure on the farm in the winter season
was present in 60% of the models, the number of broiler houses was
present in 50%, and storing manure on the farm at any time of year was
present in one-third of the models. An all-in-all-out policy at the
farm level (i.e. the practice of shipping all flocks on the farm within
the span of a few days, with all houses remaining empty for a period of
time) was not a significant predictor in any of the models. The
direction of association was inconsistent for the presence of other
commercial poultry on the farm and spreading manure on the farm in the
summer season, and the statistical significance of the former was also
inconsistent among models. In general, the models that employed a
backward elimination approach had slightly smaller AIC's and a larger
number of significant predictors than those using a forward approach.
Backward selection has an advantage over forward and stepwise selection
procedures in that negatively confounded sets of predictors are less
likely to be omitted from the model [32]. Thus, more emphasis could be placed on variables identified as significant in the backward-type models.
An increased risk of Campylobacter was associated with
increasing median flock size on the farms. For example, as the average
flock size increased by 5,000 birds, the risk of Campylobacter colonization increased by approximately 57% to 92% (i.e. 1.57 to 1.92 times). Our findings are in contrast with several studies [22-24,26] that utilized multivariable logistic regression at the flock level, in which an association between flock size and Campylobacter status
was not found. In a one-year study of 18 Swedish broiler farms,
infection risk increased when the flock size was more than 25,000 birds
[12].
However, the authors noted that since only univariable associations
were examined, their conclusions may have been confounded by farm size
and management practices. To our knowledge, ours is the first study to
examine the effect of the average flock size on the farm on the risk of
Campylobacter colonization. It has been suggested that larger
flocks require more water, feed, litter, air and personnel, all
possible sources of the bacteria [12]. Thus, in our study, increasing median flock size may be a surrogate for many other factors.
In our study, an official (municipal) water source was one in which
the water was tested regularly for coliform bacteria by the
municipality and treated if necessary, and an official treated water
source was one in which the water was treated consistently with either
ultraviolet (UV) light or a heat-cool method at the municipal level. We
found that farms using official water sources had approximately
one-third to half the risk of Campylobacter than farms using
non-official untreated sources (the referent group). Similarly, farms
using official treated water sources had roughly one-third the risk.
These findings suggest that some flocks may have been exposed through
contaminated water, as water has been identified as a suitable
reservoir and medium for Campylobacter spp. [33]. Several studies [23,34-36] have found that there was no association between the occurrence of Campylobacter in
flocks supplied with municipal (public) water compared to those
supplied with well (private) water, however, in those studies, there
was no distinction between the use of treated and untreated water
sources. We found that farms using a non-official UV-treated water
supply did not have a significantly different risk of Campylobacter than
farms using non-official untreated water at the 5% level of
significance, although in one model, non-official treated water did
have a protective effect at a 10% level. Some researchers [25,26] have found that water disinfection had a protective effect on the colonization of broilers with Campylobacter, although others [24,36,37]
have not found such an association. The small number of farms using
non-official treated water in our study, combined with potential
confounding by other factors, may account for the wide range in
p-values for this variable. Our results suggest that the use of
municipal water (both treated and untreated) reduces the risk of Campylobacter colonization
of broiler flocks, and that some potential also exists for decreasing
risk through the practice of treating non-official water sources,
depending in part on other management practices on the farm. It is
possible that there may be other, more indirect factors contributing to
the risk of colonization, such as animal density in the region. In
addition, there may be complex relationships between access of
livestock to the water source, type of water source (drilled versus
upcoming wells), and the method of water treatment (UV versus
heat-cool) that were not adequately addressed in this study. Dissection
of these inter-relationships would require a study in a country or
region with a larger number of farms.
The presence of other domestic livestock on the farm was associated with a decreased risk of Campylobacter colonization.
Similar results were obtained when we assessed the effect of the
presence of cattle, rather than the presence of other domestic
livestock in general. These findings were unexpected and inconsistent
with other studies as it has been suggested that other domestic
livestock species (especially cattle) may act as reservoirs that
potentially contaminate the farm environment thereby providing a
continual source of bacteria to the birds [13]. Several studies have shown that the presence of other animals on the farm (pigs, cattle, sheep, or fowl other than broilers) [20], (cattle) [21], (pigs, cattle, sheep and goats, or horses) [22], (laying hens, sheep, cattle, donkeys) [23] was associated with an increased risk of Campylobacter, although one recent Canadian study did not find such an association (cattle, sheep, goats, horses and/or pigs) [24]. However, in one Norwegian study [26],
the presence of other poultry or animals at the farm was not associated
with increased colonization of flocks, rather, tending other poultry
and tending pigs prior to entering the broiler house were independently
associated with an increased risk. In our study, farms that did and did
not keep other domestic livestock were similar with respect to the
number of flocks raised and the number of houses, both surrogates of
farm size. The distance between the broiler houses and the housing for
the other livestock is quite variable among broiler farms in Iceland,
with distances ranging from immediately adjacent to approximately 900 m
apart. Additionally, consistent patterns among farms in the management
of other species (e.g. manure management, assignment of workers
dedicated to a specific species, etc.) were not observed during farm
visits, although specific questions on such management practices were
not included in our questionnaires. Our findings may reflect that
Icelandic producers that raise domestic livestock in addition to
broilers take precautions that prevent contamination of the broiler
houses, such as increased efforts at biosecurity and sanitation
practices.
An increased risk of Campylobacter was associated with increasing numbers of broiler houses on the farms. For each additional house on the farm, the risk of Campylobacter colonization
increased by approximately 6% to 14%. Although we analysed this factor
as a continuous variable, our finding is consistent with several other
studies [12,22,24,36]. There was a positive correlation (τb =
0.75, p < 0.001) between the number of houses on the farm and the
number of flocks raised on the farm. To determine if the increased risk
was indeed associated with increasing numbers of houses, rather than
just increasing numbers of flocks, we included the number of flocks as
an independent predictor in the models and found that the number of
broiler houses remained statistically significant, while the number of
flocks was not significant. Several houses on the same farm may lead to
an increased risk of Campylobacter through the introduction of the bacteria into the house from the environment [36],
possibly through the increased movement of farm workers between houses,
or difficulty in maintaining strict hygiene or biosecurity practices.
In general, broiler farm workers in Iceland are not specific to a
house. However, on farms that have both breeder and broiler houses,
workers are generally assigned to either the broiler or breeder houses
and producers take precautions with any exceptions. Each broiler house
has its own set of boots and clothing, and in most cases, there is a
strict separation and physical barrier between the exterior personnel
entry area (for removal of outside boots and clothing), and the inside
clean area with dedicated broiler house boots, coveralls, and hand wash
and disinfectant. However, given the increased risk associated with
increasing numbers of houses, for new broiler farms, consideration
should be given to limiting the number of houses built.
The practice of storing manure on the farm was associated with a
decreased risk of colonization and was an unexpected finding. We
considered that this protective effect may be a result of producers
storing manure when there was not enough space to spread it in the
immediate vicinity, however, a brief exploration of the interaction
between manure storing and spreading showed that the two factors were
independent (regardless of modelling approach). One possible theory for
our finding is that manure stored in large piles (as is the practice in
Iceland) may be subjected to a form of composting or fermentation,
which may be detrimental to the survival of the organism. By contrast,
spreading manure on fields in the winter season was associated with an
increased risk of Campylobacter colonization, although it is
unclear how this practice increases risk. The effect of spreading
manure on fields in the summer season varied depending on the model. In
the automated backward stepwise model, multicollinearity was a problem,
thus, the protective effect may be a spurious result because of its
strong positive association with spreading manure in the winter. There
is very little information about these predictors in the literature,
and it is uncertain whether these practices are unique to Iceland. In
Senegalese broiler flocks, an elevated risk of Campylobacter colonization
was associated with manure disposal inside the farm compared to
disposal outside the farm, presumably through continual contamination
of the environment [23],
although the nature of disposal was not stated. Similar to our
findings, in Québec, Canada, the presence of a manure heap ≤200 m from
the broiler house (versus > 200 m) was associated with a decreased
risk of colonization, although the authors considered that this
unexpected finding was the result of confounding by farm size [24]. Analysis of these risk factors in future studies, and studies that evaluate the survival of Campylobacter in
manure under various environmental conditions, may substantially
improve our understanding of the relationship between the farm
environment and Campylobacter in broiler flocks.
A limitation of automated variable selection procedures is the
potential for inclusion of strongly correlated variables in the model.
In the automated backward stepwise model, the predictors "manure spread
on fields in summer season" and "manure spread on fields in winter
season" were both retained. The standard errors for these variables
were slightly inflated (0.8 in this model compared to approximately 0.3
in other models) as a result of multicollinearity, therefore, the
coefficients must not be over-interpreted. Notwithstanding this, these
risk factors were significant in other models suggesting their
importance in predicting the risk of Campylobacter on broiler farms in Iceland.
A second limitation of automated variable selection procedures is
the inability to identify and evaluate potential confounding variables.
In both automated forward models, the presence of other commercial
poultry on the farm was associated with an increased risk of Campylobacter colonization. This finding is in agreement with one study [12], although other researchers [26,34,36]
have not found an association. However, in both manual selection
models, the presence of other commercial poultry was shown to be a
confounder for most of the other predictors (including number of
houses, farm water source, manure spreading and storing practices, and
the presence of other livestock), and this accounts for the discrepancy
between models. Sampling of sexually immature and parent breeder flocks
in Iceland between May and July 2000, showed that up to 72% of faecal
samples were positive for Campylobacter spp. [15],
suggesting the potential for contamination of the farm environment from
these other poultry. Our results show that after controlling for other
farm-level factors, keeping other commercial poultry on the farm is not
associated with the colonization of broiler flocks with Campylobacter in
Iceland. However, in our classification of other poultry, we did not
differentiate between those farms that raised turkey flocks and broiler
flocks alternatively in the same house (with full cleaning and
disinfection between flocks), from those farms that also have year
round permanent breeder or egg layer flocks. With few exceptions, the
latter tend to be constantly heavily contaminated Campylobacter reservoirs
(based on sampling results of the on-going Icelandic surveillance
program). Future studies should carefully classify other poultry on the
farm in order to fully assess their impact on the risk of colonization
of broiler flocks.
The variable "farm has an all-in-all-out policy" changed on one farm
during the study. Since less than 80% of the flocks on this farm were
subjected to this management practice, we deemed that the farm did not
use an all-in-all-out system. In order to assess what effect this might
have had on our models, we re-analysed the data using a repeated
measures approach and found that the results were not affected. The
repeated measures approach allowed the predictor to vary for different
flocks raised on the same farm (i.e. flocks raised in the early part of
the study were subjected to an all-in-all-out system, whereas, those in
the latter part were not), and adjusted the standard errors to account
for intragroup correlation. Regardless of statistical approach, this
variable was one of the first to be removed in all of the backward
elimination procedures and was not eligible for addition in any of the
forward selection methods. Thus, in the Icelandic broiler industry, an
all-in-all-out policy on the farm does not appear to be associated with
Campylobacter colonization during the summer season. One
possible explanation for this finding may be related to the changes in
the broiler industry that took place following the epidemic in 1999 and
the implicated role of fresh broiler chicken products. Broiler
producers came under much pressure to reduce flock prevalence. A major
emphasis was placed on heightened strict biosecurity rules on broiler
farms, thorough cleaning and disinfection of houses between flocks, and
pest control. Rigorous multi-step cleaning and disinfection of the live
haul crates and trucks was also initiated. These initiatives began
early in 2000. Freezing of products from all flock lots found positive
on pre-slaughter sampling, and the price penalty to the producer for
positive flock lots, ensured continued producer motivation to maintain
high standards. This may have reduced the otherwise expected importance
of an all-in-all-out system.
Fifteen percent of the farms in our study were excluded from the
analysis due to missing data for one or more variables. In order to
assess what effect this might have had on our results, we re-analysed
the data using all 33 farms, excluding the four variables with missing
data (an all-in-all-out policy, manure spreading in the summer season,
manure spreading in the winter season, and manure storing). We found
that, whether we used 33 farms or 28 farms in our models, our estimates
for other domestic livestock on the farm, farm water source, and median
flock size were consistent. However, when we used 33 farms, the
presence of other commercial poultry and the number of houses did not
remain in any of the backward elimination models. It was evident that
there was confounding between the number of houses, the presence of
other poultry, and manure spreading & storing practices on the
farm. Therefore, by including manure management practices (and hence
analysing data from fewer farms), we likely have better estimates for
these potentially important risk factors for flock colonization at the
farm level, and the impact of other variables appears stable.
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