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Copyright 2011
Agriculture, Food and Analytical Bacteriology

Survival of Salmonella in Organic and Conventional Broiler Feed as

Affected by Temperature and Water Activity
A. Petkar1, W. Q. Alali1*, M. A. Harrison1,2, and L. R. Beuchat1
Center for Food Safety and Department of Food Science and Technology,
University of Georgia, Griffin, Georgia 30223-1797
2
Athens, Georgia 30602-2610

ABSTRACT
The objective of this study was to compare the ability of Salmonella to survive in organic and conventional broiler feeds as affected by temperature (11, 25, and 38C), water activity (aw 0.75, 0.55, and 0.43) and
storage time (up to 80 days). Feeds were inoculated with a mixture of five Salmonella serotypes at high
and low populations (6 and 3 log CFU/g, respectively), and populations and presence (by enrichment) were
monitored over time. Although the number of Salmonella in organic feed for the majority of temperatureby-aw combinations was significantly lower (P 0.05) compared to the number in conventional feed over
the 80-day storage period, differences in mean populations were less than 1 log CFU/g. The odds-ratio
(OR) for presence of Salmonella was significantly higher (P 0.05) in conventional feed than in organic feed
containing high and low inocula (OR = 4.76 and 2.92, respectively). Based on these findings, we generally
conclude that there were no biologically significant differences in survival of Salmonella in organic and
conventional poultry feeds.
Keywords: Salmonella, conventional poultry feed, organic poultry feed, storage temperature, water activity, aw

Agric. Food Anal. Bacteriol. 1: 175-185, 2011

INTRODUCTION
Salmonella is one of the most common pathogens known to cause foodborne disease in the United States and worldwide. According to Scallan et al.
(2011), an estimated 1.03 million people suffer from
Correspondence: Walid Q. Alali , walali@uga.edu
Tel: +1 - 770-467-6066 Fax: +1-770-229-3216

salmonellosis annually in the U.S. Nontyphoidal Salmonella is considered to be the leading cause of
foodborne illness-related hospitalizations and death
in the U.S. Poultry and poultry products are considered to be an important source of Salmonella (Tauxe,
1991; Bryan and Doyle, 1995) and contaminated feed
is considered to be one of the main sources of Salmonella infection in broiler birds (Jones et al., 1991;
Maciorowski et al., 2004).

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175

The presence of Salmonella in poultry feed as

well as feed ingredients such as grain, oilseed meal,
feathers, fishmeal, and meat by-products has been
documented (Williams 1981a; Cox et al., 1983; Stuart 1984; Veldman et al., 1995). After contaminated
feed is consumed by broilers, Salmonella can multiply in the gastrointestinal tract of the bird and potentially be shed in the feces during grow out (Cason
et al. 1994). Several intervention methods, including
heating and pelleting at 70C and 90C, irradiation
(gamma rays), and addition of chemicals (organic acids, organic salts, formaldehyde, and bacterial membrane disruptors such as terpenes and essential oils)
have been applied to poultry feed and feed ingredi-

survive in USDA-certified organic broiler feed versus

conventional broiler feed stored at different temperatures and aw over an 80-day period.

ents to control Salmonella (Wilder, 1969; Hinton and

Linton, 1988). Despite the use of these interventions,
poultry feed can be recontaminated with Salmonella
post-production, e.g., during storage at the feed
mill, transportation, and storage at the farm (Hinton
and Linton, 1988).
Storage of poultry feed under various environmental conditions may influence the survival and
growth of Salmonella. Reports from several studies
show that survival of Salmonella is influenced by factors such as the presence of antimicrobials, moisture
content, and storage temperature (Himathongkham
et al., 1996; Halls and Tallentire 1978; Furuta et al.,
1980; McCapes et al., 1989). Juven et al. (1984) reported that survival of Salmonella is greater at a
water activity (aw) of 0.43 than at 0.75. The survival
and heat resistance of Salmonella in poultry feed has
been reported to be inversely related to moisture
content and relative humidity (% relative humidity =
aw x 100), except at a moisture content that allows
growth (Liu et al., 1969; Carlson and Snoeyenbos,
1970; Juven et al., 1984).
In a recent study, it was found that conventional
poultry feed was contaminated with Salmonella,
whereas USDA-certified organic feed was Salmonella-free (Alali et al., 2010). It is unclear whether the

at 37C. A loopful of TSB culture of each serotype was

streaked on a double-layered agar medium consisting of tryptic soy agar (TSA) (Difco, BD) and xylose
lysine tergitol (XLT4) agar (Difco, BD), and incubated
at 37C for 24 h. The TSA-XLT4 agar was prepared
as described in Kang and Fung (2000). This agar has
been used for direct plating to support recovery and
enumeration of injured Salmonella. The agar is composed of a bottom layer of XLT4 agar as a selective
medium and thin top layer of nonselective TSA.

organic feed was contaminated with Salmonella

which subsequently did not survive or was present
at a level below the limit of detection, or whether
the feed was not contaminated. The objective of this
study was to determine the ability of Salmonella to
176

MATERIALS AND METHODS

Salmonella serotypes
Five Salmonella enterica serotypes were obtained
from the Poultry Diagnostic Research Center, University of Georgia, Athens, GA. These serotypes were S.
Typhimurium, S. Heidelberg, S. Enteritidis, S. Montevideo, and S. Gaminara. All serotypes were grown in
tryptic soy broth (TSB) (Difco, BD; Sparks, MD) for 24 h

Salmonella inoculum
To prepare the Salmonella inoculum, 6-ml quantities of nutrient broth (NB) (Difco, BD) were inoculated
with cells from single colonies of Salmonella formed
on TSA-XLT4 agar plates incubated at 37C for 24 h.
Each serotype was cultured separately. The NB inoculum concentration was adjusted to approximately 8
log CFU/ml using a spectrophotometer (Spectronic
20; Bausch and Lomb, Rochester, NY) at an optical density of 0.5 to 0.6, as described by Kaiser et al.
(2002). This suspension was used to prepare a highpopulation dry chalk powder inoculum. Ten-fold serial dilutions were made in Nutrient Agar (NA) to prepare a low-population chalk inoculum.

Preparation of dry chalk inocula

Chalk was used as a carrier for preparing a dry Salmonella inoculum (Okelo et al., 2008). Prior to use,

Agric. Food Anal. Bacteriol. AFABjournal.com Vol. 1, Issue 2 - November 2011

Table 1. Composition of conventional and

organic broiler-grower feeds.

Corn meal

72.79

65.80

Soybean meal

15.80

23.77

Alfalfa meal (dehydrated)

1.20

Poultry meat & bone meal

6.00

Fat

2.70

5.00

Dicalcium phosphate

0.20

1.24

Limestone

0.66

1.55

Salt

0.39

0.31

Methionine

0.29

0.23

Crayola chalk (Code #51-0320, Binney and Smith,

Easton, PA) was sterilized by autoclaving. The chalk
did not possess antimicrobial properties. The pH of
the chalk was 7.0, as measured by a pH meter (Fisher
Scientific, Pittsburgh, PA). Chalk (4.3-g pieces) was
submerged in the five-serotype mixture of high- or
low-population NB suspension for 12 h at 37C, then
placed in sterile petri dishes and dried for 72 h at
37C. The inoculated, dried chalk was pulverized using a food processor (Hamilton Beach Food Processor, model 70590, type FP 11, Southern Pines, NC) in a
laminar air flow chamber to obtain powdered inocula
containing Salmonella at approximately 7 log CFU/g
(high inoculum) and 4 log CFU/g (low inoculum) of

Vitamin premix2

0.25

0.12

chalk. The aw of chalk powder inocula was 0.22.

0.65

0.70

Trace mineral premix

0.08

0.08

Coban4

0.05

BMD5

0.05

L-Threonine

0.07

TBCC5

0.02

Conventional Organic
feed (%)
feed (%)6

Ingredient

Lysine
3

Vitamin mix contained the following: vitamin A, vitamin

D3, vitamin E, vitamin B12, riboflavin, niacin, d-Pantothenic
acid, choline, menadione, folic acid, thiamine, pyridoxine,
biotin, and ethoxyquin.
1

Trace mineral mix contained the following: calcium, iron,

magnesium, manganese, zinc, copper, iodine, selenium.
2

Coban: coccidiostat

Enumeration of Salmonella in dry chalk

inocula
Enumeration of viable Salmonella was done by
adding 10 g of high- or low-population chalk inoculum to 90 ml of phosphate buffered saline (PBS),
vigorously shaking the suspension, and making 10fold serial dilutions in PBS. Samples (0.1 ml) were
surface plated in duplicate on TSA-XLT4 agar plates
and incubated at 37C for 24 h before colonies were
counted.

BMD: bacitracin methylene disalycilate

TBCC: tribaic copper chloride

Specific ingredients listed for organic broiler feed: monosodium phosphate, organic kelp meal, diatomaceous
earth, , ferrous sulfate, organic apple cider vinegar, , zinc
sulfate, , organic potato starch, organic dehydrated eggs,
organic dried tomato pomace, organic dried whole milk,
organic linseed meal, organic aloe vera gel concentrate,
organic soybean oil, organic oat flour, lecithin, organic
wheat middlings, organic sugar, potassium chloride, attapulgite clay, pyridoxine hydrochloride, folic acid, ferric
choline citrate complex, zinc choline citrate complex, carotene, ascorbic acid, yeast culture, cobalt sulfate, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium
thermophilum, Enterococcus faecium, organic sources of
(cayenne pepper, peppermint, garlic, parsley, dandelion
root extract, elder flowers, dandelion extract, ginger extract, German chamomile, lemon grass extract, thyme,
sweet fennel extract, sweet basil, sage, cloves), and natural
tocopherols.
6

Feeds used
Conventional pelleted broiler feed formulated for
grower birds (Table 1) was purchased from two conventional poultry companies (companies A and B).
The formulations listed for these feeds were similar.
Organic mash feed formulated for grower birds (Table 1) was obtained from two organic poultry companies (companies C and D). Prior to inoculation,
feed samples were tested (as described later under
Salmonella analysis-selective enrichment) to ensure
that they were negative for Salmonella.

Preparation of feeds with desired aw

Saturated salt solutions (potassium carbonate,
sodium bromide, and sodium chloride) were placed

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177

inside each cabinet to attain the desired aw (Rockland, 1960). Three cabinets at three different relative
humidities were each kept at three different temperatures (11, 25, and 38C). Conventional and organic
feeds were stored inside secador desiccator cabinets
(Structure Probe, Inc., West Chester, PA) for 3 weeks
to reach target aw values of approximately 0.43, 0.55
and 0.75 prior to inoculating with Salmonella. The aw
of feeds was measured over time using an Aqualabaw meter (Decagon Devices, Inc., Pullman, WA).

(0.1 ml) from each dilution (10-1 to 10-3) were spread

plated on TSA-XLT4 agar plates. After incubating
plates for 24 h at 37C, colonies presumptive for
Salmonella were enumerated. For the low-inoculum
samples, the procedure for enumerating Salmonella
was similar with the exception that suspensions were
not diluted. Undiluted LB/feed homogenates were
surface-plated on TSA-XLT4 agar. The LB/feed suspensions were incubated for 24 h at 37C.

Selective enrichment

Experimental design
All experiments were replicated twice. Feeds from
companies A and C (conventional and organic, respectively) were used in the first trial, whereas feeds
from companies B and D were used in the second
trial. A 3 x 3 factorial design was used to conduct
the study. For each feed containing either high- or
low inoculum, three storage temperatures (11, 25,
and 38C) and three aw levels (0.75, 0.55 and 0.43)
were tested to determine their effects on survival of
Salmonella in conventional and organic broiler feeds
over a 80-day period. For each temperature/aw combination, triplicate 9-g samples of conventional and
organic feed in sterile glass test tubes were inoculated with 1 g of powdered chalk inoculum to obtain
populations of approximately 6 log (high) and 3 log
(low) CFU/g. The inoculated feeds were mixed, deposited in tubes, and placed inside the cabinets.

When direct plating of diluted feed samples was

negative for Salmonella, 1 ml and 0.1 ml of preenriched LB/feed mixture were inoculated into 10 ml
of tetrathionate (TT) broth (Difco, BD) and 10 ml of
Rappaport Vassiliadis broth (RV) broth (Difco, BD),
respectively. After incubating broths for 24 h at 35C
(TT broth) and 42C (RV broth), a loopful of the culture was streaked on TSA-XLT4 agar. After incubating plates for 24 h at 37C, cells from colonies presumptive for Salmonella were inoculated into triple
sugar iron (TSI) agar slants (Difco, BD), incubated
with caps loosened at 35C for 18 to 24 h, and examined for carbohydrate fermentation, gas production,
and hydrogen sulfide production. Based on these
observations, feed samples were judged as positive
or negative for Salmonella.

Statistical analysis

Triplicate 10-g samples of inoculated feed stored

for 0, 3, 7, 14, 21, 28, 35, 50, 65, and 80 days at each
temperature/aw combination were analyzed for
populations and presence (by enrichment) of Salmonella. For the high-inoculum samples, 10 g were
suspended in a 90 ml of Luria Bertani (LB) (Sigma Al-

Populations of Salmonella (log CFU/g) determined by direct plating samples of conventional and
organic broiler feeds were compared among each
temperature/aw combination, by inoculum level, and
at each storage day using repeated measures of
analysis of variance ANOVA in General Linear Model
(GLM) in SAS software version 9.1.3 (GLM procedure,
SAS Inst., Inc., Cary, NC). Salmonella counts were
logarithmically transformed by use of log base 10 to

drich Corp., St. Louis, MO) broth in a 250-ml flask,

followed by shaking for 1 h on a rotary shaker (New
Brunswick Scientific, Edison, NJ). Ten-fold serial dilutions of the suspension were made in sterile microcentrifuge tubes containing 0.9 ml of (PBS). Aliquots

approximate normality. Data from the two replicate

experiments were tested using Levenes test for homogeneity of variances. The variances between the
two replicate experiments were not significantly different (P > 0.05). Based on these findings, data from

Analysis of feeds for Salmonella

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Table 2. Mean Salmonella populations (log10 CFU/g) in conventional and organic poultry feed initially containing high or low inoculum and at different temperature and aw combinations over an
80-day storage period.
Salmonella (log CFU/g)
Inoculum level

aw

Temp (C )

Conventional feed

Organic feed

P-valuea

High

0.43

11

6.17

5.93

<0.001

25

5.29

5.05

0.140

38

2.69

2.52

0.600

11

6.21

5.93

0.055

25

5.55

5.09

0.030

38

2.86

2.44

0.150

11

6.31

6.16

0.030

25

5.67

4.79

0.001

38

2.44

2.22

0.380

11

4.29

4.12

0.020

25

3.26

2.64

<0.001

38

1.10

0.80

0.009

11

4.58

4.24

<0.001

25

3.89

3.15

<0.001

38

1.39

1.06

0.011

11

4.43

4.08

<0.001

25

2.96

1.86

<0.001

38

0.93

0.81

0.003

0.55

0.75

Low

0.43

0.55

0.75

P-values were considered significant at 0.05 at different temperatures and aw.

both replicate trials were pooled to obtain a set of

six observations for each sampling day. In addition,
the proportion of samples positive for Salmonella
was compared among each temperature-by-aw combination, by inoculum level, and at each sampling
time using a GLM with a binomial distribution and a
logit link in SAS (GENMOD procedure). The reported odds ratios (OR) from GLM model was comparing
the odds of Salmonella in one type of feed a group
to the odds of Salmonella in the other type of feed
(Dohoo et al., 2003). A sample was considered positive if direct plating was positive or if enrichment was
positive when direct plating was negative.

RESULTS
All uninoculated feed samples were negative for
Salmonella. Salmonella populations in inoculated

conventional and organic broiler feeds were determined over an 80-day period. Although the differences between populations in the two types of feeds
held at various temperatures/aw conditions were
small (generally < 1 log CFU/g), these differences
were statistically significant (P 0.05) within both
inoculum levels. For the feeds containing a high
inoculum, mean log CFU of Salmonella/g over the
80-day storage period were 4.71 0.09 and 4.36
0.09 for conventional and organic feed, respectively.
For feeds containing the low inoculum, the mean log
CFU of Salmonella/g were 2.88 0.08 and 2.38
0.08 for conventional and organic feed, respectively.
We considered differences in Salmonella populations between organic and conventional feeds to be
biologically meaningful if they were significantly different and >1 log CFU/g.
At day 0, the mean populations of Salmonella re-

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179

Figure. 1. Mean number of Salmonella (log CFU/g) in conventional and organic poultry feeds [high
inoculum level (106 CFU/g)] over a storage period of 80 days at various temperature/aw conditions:
A to C = 11oC, aw 0.43, 0.55, and 0.75; D to F = 25oC, aw 0.43, 0.55, and 0.75; and G to I = 38oC, aw
0.43, 0.55, and 0.75. Each data point represents the mean of values from three replicate samples
for two experiments (n = 6) per treatment.
0.55

0.75

11C

0.43

38C

25C

covered from inoculated conventional and organic

feeds containing high and low inocula levels were
approximately 6 and 3 log CFU/g, respectively, indicating an approximate 1-log CFU/g reduction based
on the number of Salmonella added to the feeds via
chalk inocula. The effects of different temperature/
aw combinations by feed type and inoculum level on
survival of Salmonella over an 80-day storage period
are shown in Table 2. The mean Salmonella populations in organic and conventional feeds initially con-

0.75) stored at 25C for 50 and 65 days (Fig. 1, F).

There were significant differences (> 1 log CFU/g)
in low-inoculum feeds stored at 25C and aw 0.43,
0.55, and 0.75 during the study period (Fig. 2, D-F).
Regardless of inoculum level, Salmonella in organic
and conventional feeds decreased to an undetectable level (2 log CFU/g by direct plating; 1 CFU/10 g
by enrichment) in feeds stored at 38C, regardless of
the aw (Fig. 1, G-I; Fig. 2, G-I).
Considering all temperature/aw combinations, the

taining high and low inocula and stored at different

temperature/aw combinations for up to 80 days are
shown in Figures 1 and 2, respectively. There were
statistically significant differences (P 0.05) and >
1 log CFU/g difference in high-inoculum feeds (aw

OR for the presence of Salmonella in conventional

feed was significantly (P 0.05) higher than that in
organic feed containing a high inoculum (OR = 4.76
[95% confidence interval {CI}], 2.66 to 22.57). The OR
for presence of Salmonella in low-inoculum conven-

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tional feed was significantly higher (P 0.05) than for

organic feed (OR = 2.92 [95% CI, 2.16 to 4.53]). The
GLM model comparing Salmonella populations in
conventional feed versus organic feed (as the reference category) failed to converge due to the sparse
amount of data (no or few positives) for feed stored
at most temperature/aw combinations, regardless of
the inoculum level. The percentage of Salmonellapositive conventional feed samples containing a
high inoculum level decreased by 50% at the end of
80-day storage period as compared to 43% in organic feed (Fig. 3). In contrast, at a low inoculum level,
the percentage of Salmonella-positive conventional
feed samples decreased 58% by the end of the 80-

ter and fall, 25C in spring, and 38C in summer) in

southeastern U.S. These conditions were selected
to simulate environmental conditions for handling
and storage of poultry feed along the productionto-consumption chain. Furthermore, to investigate
differences in contamination levels, poultry feeds
were inoculated with high and low populations of
Salmonella. Since poultry feeds are generally stored
for less than 8 to 10 weeks from production to consumption, the period of 80 days was chosen to evaluate the survivability of Salmonella.
Although most of the differences in mean Salmonella populations between the two types of feed, at
each temperature/aw combination, and by inoculum

day storage period as compared to 40% in organic

feed (Fig. 3).

The ability of Salmonella to survive in organic

versus conventional broiler feed as affected by storage temperature and aw has not been reported, although in the past decades, poultry feed has been
considered to be an important vehicle for transmission of Salmonella to broiler birds. Salmonella has
been detected in poultry feed and feed ingredients (Williams, 1981a; Williams, 1981 b; Cox et al.,
1983; Stuart, 1984; Veldman et al., 1995). In our experiments, a dry inoculation technique rather than
a liquid-suspension inoculation was used to distribute Salmonella in feeds. The dry inoculum did not
substantially alter the aw of the feed. Compared to
a wet inoculum, distribution of Salmonella from dry
inocula is homogenously distributed and changes
in background microbiota are expected to be minimum (Hoffmans and Fung, 1993). In addition, dry
inoculation more closely mimics contamination that
might occur via dust and other dry materials in growout facilities.
We evaluated three temperatures and three aw

population were statistically significant (Table 2),

the numerical differences were too small (<1.0 log
CFU/g) to be considered biologically meaningful.
The proportion of samples positive for Salmonella
was significantly higher in conventional feed compared to organic feed for both inoculum levels over
all temperature/aw combinations. Differences in composition of organic and conventional feeds (Table 1)
did not appear to impact the survival of Salmonella;
however, feed composition may have impacted the
percentage of samples positive for Salmonella in the
two types of feed. A possible explanation for this difference may be attributable to the absence of animal protein meals (blood and bone) and presence
of natural antimicrobials such as garlic, clove, ginger,
and basil in organic feed. Leuschner and Zamparini
(2002) tested the effect of different natural antimicrobials such as garlic, ginger, mustard, and cloves on
growth and survival of Escherichia coli O157 and S.
enterica serotype Enteritidis in broth model systems.
Garlic and clove showed bacteriostatic and bacteriocidal effects on both foodborne pathogens. Clove
was found to be more effective than garlic. Mustard
and ginger also exhibited bacteriostatic activities
against both bacteria.
When examining survival of Salmonella as affect-

values covering ranges existing in storage facilities

at feed mills, during transportation, and on-farm
during different seasons. Poultry feeds are marketed
at aw ranging from about 0.45 to 0.75 and exposed
to various storage temperatures, (e.g., 11C in win-

ed by storage time (Figs. 1 and 2), a few differences

>1 log CFU/g were observed, mainly in feed containing a low inoculum and stored at 25C. Salmonella
in both high- and low-inoculum feeds stored at 38C
decreased to populations below the limit for detection

DISCUSSION

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Figure. 2. Mean number of Salmonella (log CFU/g) in conventional and organic poultry feeds [low
inoculum level (103 CFU/g)] over a storage period of 80 days at various temperature/aw conditions:
A to C = 11oC, aw 0.43, 0.55, and 0.75; D to F = 25oC, aw 0.43, 0.55, and 0.75; and G to I = 38oC, aw
0.43, 0.55, and 0.75. Each data point represents the mean of values from three replicate samples
for two experiments (n = 6) per treatment.
0.55

0.75

11C

0.43

38C

25C

by direct plating (2 log CFU/g). Similar observations in

conventional poultry feed were reported by Williams
and Benson (1978), where S. Typhimurium survived
much longer at 11 and 25C than at 38C. In our study,
in feed containing a high inoculum, the mean number of Salmonella recovered from both types of feed
ranged from 4 to 5 log CFU/g after storage for 80 days
at 11 and 25C, regardless of aw. This observation con-

of cells, thus immediately lowering the number of viable Salmonella as compared to what may occur in the
feed to which a dry inoculum is added. The inoculation
procedure and aw of feeds have been reported to affect the survivability of Salmonella (Liu et al., 1969; Carlson and Snoeyenbos, 1970; Juven et al., 1984).
Previous studies show that Salmonella does not
grow but survives well in low-aw foods such as peanut

curs with the findings of Davies and Wray (1996) showing that S. Typhimurium declined to 3 log CFU/g of
conventional poultry feed over a period of 3 months.
These researchers inoculated feed using a suspension
of Salmonella that may have resulted in osmotic shock

butter, infant formula, cereals, and dry aniseed (GMA,

2009). These and other low-aw products have been
associated with outbreaks of salmonellosis. Investigations suggest that factors such as poor sanitation
practices, poor equipment design, improper mainte-

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Agric. Food Anal. Bacteriol. AFABjournal.com Vol. 1, Issue 2 - November 2011

Figure. 3. Percentage of Salmonella-positive

samples in conventional and organic poultry
feeds initially containing a high- and low inocula and stored up to 80 days.

and 0.55, Salmonella survived at higher levels in both

types of feed as compared to survival at aw 0.75. Reductions were more rapid as the storage temperature was
increased.

ACKNOWLEDGEMENTS
We thank Dr. John Maurer from Poultry Diagnostic Research Center, University of Georgia, Athens,
GA for providing the Salmonella isolates used in this
study. We also thank Rebekah Turk for her technical
assistance in the laboratory.

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