C 2015 Poultry Science Association Inc.
Effects of dietary chlorine dioxide on growth
performance, intestinal and excreta microbiology,
and odorous gas emissions from broiler excreta
Sonia Tabasum Ahmed, Gyeongil Kim, Md. Manirul Islam, Hong-Seok Mun,
A. B. M. Rubayet Bostami, and Chul-Ju Yang1
Department of Animal Science and Technology, Sunchon National University,
255 Jungangno, Suncheon, Jeonnam 540-950, Republic of Korea
Primary Audience: Veterinarians, Poultry Farmers, Poultry Scientists, Nutritionists
SUMMARY
Chlorine dioxide (ClO2 ) is a powerful biocide that has long been used commercially to
control microbial activity in various sectors. This study was conducted to determine the effectiveness of dietary ClO2 on growth performance, intestinal and excreta microorganisms, and
the emission of odorous gas from broiler excreta. A total of 120 one-day-old broiler chicks
were provided with experimental diets including ClO2 at 0, 0.05, or 0.1% of the diet at random.
Dietary ClO2 (0.05 and 0.1%) resulted in a significant reduction in feed intake and feed conversion ratio without affecting the growth rate of the broilers. Dietary ClO2 (0.05 and 0.1%)
did not affect the Lactobacillus, Bacillus, and Salmonella concentrations in the ileal digesta;
however, it significantly increased the level of yeast and mold. In contrast, dietary ClO2 at 0.1%
significantly decreased the number of ileal E. coli. In the cecal digesta, the Bacillus, E. coli and
Salmonella CFUs were reduced significantly in response to 0.05 and 0.1% ClO2 . Both levels of
ClO2 significantly reduced the concentrations of E. coli and Salmonella in broiler excreta by d
21, whereas significant reduction was observed only in response to 0.1% ClO2 supplementation
by d 35. Dietary supplementation with 0.1% ClO2 resulted in significant reductions in excreta
pH at 6, 12, and 48 h of incubation relative to the non-supplemented group. Fecal ammonia
emission was significantly higher at 48 h in response to 0.1% ClO2 supplementation. In contrast,
significantly lower emissions of hydrogen sulfide (3, 6, 12, and 24 h), sulfur dioxide (24 and
48 h), and mercaptans (0, 3, and 48 h) were recorded in response to dietary ClO2 supplementation. Overall, reduction of intestinal and excreta pathogenic microorganisms together with lower
emissions of sulfur-containing odorous gas from broiler excreta were observed in response to
dietary supplementation with 0.1% ClO2 without affecting the growth rate.
Key words: broilers, chlorine dioxide, microbiota, pH, excreta odorous gas
2015 J. Appl. Poult. Res. 24:502–510
http://dx.doi.org/10.3382/japr/pfv058
DESCRIPTION OF PROBLEM
Broilers have a diversified intestinal microbial ecology that includes beneficial and harm1
Corresponding author: [email protected]
ful bacteria that greatly influence their health
status through various functional roles in terms
of nutrition, immunity, detoxification, and other
physiological systems [1] When excreted with
feces, these microorganisms also play a major
role in the emission of odorous volatile organic
AHMED ET AL.: CHLORINE DIOXIDE IN BROILERS
compounds from animal manure through degradation of fecal substances [2]. Therefore, stabilizing the intestinal ecosystem of birds by enhancing the growth of beneficial bacteria and
reducing the number of pathogenic bacteria is
of crucial importance. It has been demonstrated
that manipulation of gut microflora through diet
is an efficient tool for improving growth performance and feed efficiency of domestic animals.
The use of antibiotic growth promoter in animal
diets is assumed to influence gut health stabilization. However, owing to the development of bacterial resistance against antimicrobial substances
and the ban of most antibiotic feed additives,
there is increased interest in using alternative
antimicrobials in poultry diets.
Chlorine dioxide (ClO2 ) is a disinfectant approved by the USDA-Food Safety Inspection
Service for use as an antimicrobial chemical [3]
that has been reported to be an effective means of
reducing bacterial contamination of broiler carcasses [4], wash water [5], and surfaces [6]. It
is also considered to be an effective bactericide
that results in less formation of chlorinated organic compounds than aqueous chlorine under a
relatively wide range of pH conditions [7]. Most
importantly, bacteria do not develop resistance
against ClO2 because it reacts with biological
thiols, which play a vital role in all living organisms [8]. ClO2 kills microorganisms via direct action on the cellular membrane and through
disruption of fundamental cellular processes [9].
Barnhart et al. [10] suggested that appropriate
disinfectants could be administered via the oral
route during pre-slaughter feed withdrawal to
reduce foodborne pathogens from the gastrointestinal tract. Oral ingestion of ClO2 at levels up
500 ppm (0.05%) had no toxic effects in chickens
and pigs [11, 12] and did not influence palatability of diet or feed intake [11], although it did
show strong bactericidal and virucidal activity.
Additionally, a case study of ClO2 administered
with drinking water revealed improved feed conversion ratio in broilers with reduced mortality
[13]. However, to the best of our knowledge, no
studies have tested the effects of ClO2 administration with broiler diet. Therefore, we investigated whether supplementing broiler diets with
ClO2 powder would affect intestinal microbiota,
thereby improving nutrient utilization and feed
efficiency, and reducing odorous gas emissions
503
from broiler excreta. Specifically, we observed
the effects of two levels of ClO2 on the growth
performance, intestinal microbiota and pH, excreta microbiota and pH, and odorous gas emissions from broiler excreta.
MATERIALS AND METHODS
Birds, Management, and Diets
A total of 120 one-day-old male broiler chicks
(Ross 308) were purchased from a commercial
hatchery (Yangil Farm, Yeosu, Republic of Korea) and kept in a closed, ventilated, wire-floor
caged broiler house (80 cm long × 60 cm wide
× 40 cm high/cage) at a stoking density of
600 cm2 /bird. The chicks were randomly divided
into 3 treatment groups with 5 replicate cages of
8 birds. The cages of each treatment were distributed in such a way that they are located in
the front, middle, and back of the house. Temperature was maintained at 33◦ C for d 1 to 7,
after which it was gradually reduced to 24◦ C at a
rate of 3◦ C per week and then maintained at this
temperature until the end of the experiment. The
relative humidity was maintained at around 50%
and continuous lighting was provided throughout the experimental period. Three dietary levels
of ClO2 (0, 0.05, and 0.1% of diet) were used
in this experiment by removing equal weight of
basal diet and were fed in 2 phases: starter diet
from 0 to 21 d and finisher diet from 22 to 35 d.
As shown in Table 1, the basal diets were formulated to meet or exceed the nutrient requirements
of broilers according to the National Research
Council [14]. The use and care of birds and all
the experimental procedures were approved by
the Animal Care and Use Committee of Sunchon National University, South Korea.
Measurements and Analyses
Growth Performance Assay. Feed intake and
BW were recorded by replicate at 7, 14, 21, 28,
and 35 d of age. The ADFI, ADG, and FCR
were then calculated from these data per cage
by period and for the entire experimental period.
The mortality was recorded once observed to
adjust the FCR data.
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Table 1. Feed ingredients and analyzed chemical compositions of
broiler basal diets.
Item
Starter (0 to 21 d)
Ingredients (%, as-fed basis)
Corn grain
Soybean meal
Corn gluten
Soybean oil
Animal fats
Salt
Dicalcium phosphate
Limestone
Vitamin-mineral premix1
Choline
L-Lysine HCL (78%)
DL-Methionine
Nutrient content (% DM)
MEn (kcal/kg)
Crude protein (%)
Crude fat (%)
Crude ash (%)
Crude fiber (%)
Ca (%)
Available phosphorus (%)
Lysine (%)
Methionine (%)
57.37
26.80
5.00
2.20
4.50
0.25
2.14
0.92
0.30
0.08
0.24
0.20
3200
22.2
4.65
5.63
4.42
0.80
0.55
1.45
0.53
Finisher (22 to 35 d)
60.64
24.90
3.50
2.20
5.00
0.25
2.00
0.88
0.30
0.07
0.16
0.10
3200
20.7
2.43
5.61
3.71
0.75
0.52
1.10
0.45
1
Vitamin-mineral mixture provided the following nutrients per kilogram of
diet: vitamin A, 15,000 IU; vitamin D3, 1,500 IU; vitamin E, 20.0 mg;
vitamin K3, 0.70 mg; vitamin B12, 0.02 mg; niacin, 22.5 mg; thiamine, 5.0
mg; folic acid, 0.70 mg; pyridoxine, 1.3 mg; riboflavin, 5 mg; pantothenic
acid, 25 mg; choline chloride, 175 mg; Mn, 60 mg; Zn, 45 mg; I, 1.25
mg; Se, 0.4 mg; Cu, 10.0 mg; Fe, 72 mg; Co, 2.5 mg (Bayer Korea Ltd.,
Dongjak-Ku, Seoul, Korea).
Sample Collection and Analyses of Intestinal Microbiology and pH. At d 35, 3 birds
were selected at random from each replicate
for ileal and cecal sample collection. The selected chickens were exsanguinated by cutting
their jugular vein, after which the gastrointestinal tracts were removed from the carcasses. Next,
the ileum and 10-cm segments from the same
area of both ceca were dissected. Approximately
1 g of ileal or cecal content was aseptically collected from 3 birds per replication into 10 mL
safe-lock conical tubes (SPL Life Sciences Co.,
Ltd., Pocheon, Gyenggi-do, Korea) and diluted
to a 1:9 wt/vol ratio in sterile sodium chloride
saline (Daejung Chemical & Metals Co., Ltd.,
Siheung, Gyonggi-do, Korea), which was then
homogenized for 2 min. Serial dilutions (1:10)
were prepared, after which 20 µL diluted samples
were plated in duplicate on DifcoTM Lactobacilli
MRS (Mann, Rogosa, and Sharpe) agar to count
Lactobacillus spp., DifcoTM Mannitol-Egg YolkPolymyxin (MYP) agar to count Bacillus spp.,
DifcoTM Potato Dextrose agar to count yeast
and mold, DifcoTM MacConkey Sorbitol agar to
count Escherichia coli, and BBLTM Salmonella
Shigella agar to count Salmonella spp. (Becton,
Dickinson, and Company, Sparks, MD). Potato
dextrose agar plates were incubated for 48 h at
37◦ C, while the other agar plates were incubated
for 24 h at 37◦ C. Visible microbial colonies
were counted immediately after removal from
the incubator considering the color of colonies
(Lactobacillus spp., large white colonies; Bacillus spp., yellow colonies; yeast and mold, white
colonies; E. coli, rose to pink colonies; S. Typhimurium, colorless with black center colonies)
and expressed as log10 cfu/g.
The pH was determined by suspending approximately 0.1 g of cecal contents in 0.9 mL
of distilled water (DW) and then measuring the
AHMED ET AL.: CHLORINE DIOXIDE IN BROILERS
sample with a Uni pH testa (Trans Instruments
(S) PTE Ltd., 5 Jalan Kilang Barat, Petro Centre,
Singapore) at 25◦ C.
Sample Collection and Analyses of Excreta
Microbiology and pH. For microbial analyses,
fresh fecal samples were collected from the bottom tray of each wire-floor cage into zipper plastic bags at d 21 and 35 of the experimental period.
One g of fecal sample was weighed and diluted
to a 1:9 wt/vol ratio in sterile sodium chloride
saline, homogenized for 2 min, and then subjected to serial 10-fold dilutions. The microbial
plating, incubation, and counting methods were
the same as for intestinal digesta.
To measure the fecal pH, 4 g of fecal sample
were weighed and diluted to a 1:9 wt/vol ratio in
36 mL of DW, after which the pH was measured
directly using a Uni pH testa (Trans Instruments
(S) PTE Ltd, 5 Jalan Kilang Barat, Petro Centre,
Singapore) at 25◦ C.
Measurement of Odorous Gas Emissions
from Excreta. To collect fecal samples for excreta odorous gas emission, feces in the bottom
tray of each wire-floor cage were homogenized
and put in plastic zipper bags (d 36 of experiment). About 500 g of fecal sample from each
replicate cage were then placed in 2-liter plastic
boxes in triplicate to measure the emission of
ammonia (NH3 ), hydrogen sulfide (H2 S), sulfur
dioxide (SO2 ), and mercaptan from broiler excreta. The boxes used to measure the gas emissions were equipped with covers containing two
holes, one that was used to insert a tube with a
cap to facilitate gas measurement and the other
R
membrane
that was sealed with an Advantec
filter (pore size 1.0 µm, Toyo Roshi Kaisha Ltd.,
Otowa, Tokyo, Japan) to facilitate insertion of
fresh air to fill the negative pressure created during drawing of the headspace air. Following one
sampling at 0 h, the samples were allowed to ferment at room temperature (average 27◦ C) and
subsequent samples were collected at 3, 6, 12,
24, and 48 h. A Gastec (model AP-20) gas sampling pump (Gastec Corp., Kitagawa, Japan) and
Gastec detector tube (3 LA, 3M for NH3 ; 4 LB,
4LK for H2 S; 5 LA for SO2 ; 70L for mercaptans)
were used to measure the odorous gas emissions.
During measurement, the tube was open and 100
mL of headspace air was sampled from approximately 2.0 cm above the sample surface. The
505
concentration of odorous gases was expressed
as ppm/100 mL.
Statistical Analyses
All experimental data were analyzed in accordance with the General Linear Model Procedure established by the Statistics Analysis Systems Institute [15]. The individual cage served
as the experimental unit for performance parameters, excreta microbiota, pH, and odorous gas
emissions, whereas a group of 3 birds served as
an experimental unit for ileal and cecal microbiota and pH. Treatment means were separated
using a Student’s t test. A probability level of
P < 0.05 was considered to be statistically significant, whereas a P < 0.10 was considered to
constitute a tendency.
RESULTS AND DISCUSSION
The effects of dietary ClO2 on the growth performance of broilers are shown in Table 2. The
results showed that the BW and ADG of broilers
were unaffected, but a significant reduction in
ADFI with improved FCR was observed in response to dietary inclusion of ClO2 throughout
the experimental period (P < 0.05). In contrast
to our results, Demeckova et al. [11] reported
that oral ingestion of ClO2 at a level of 500 ppm
(0.05%) had no negative effect on feed intake or
performance of newly weaned piglets. The mortality rate of broilers was not affected by ClO2
supplementation (data not shown).
Inactivated stabilized ClO2 works well as
a bacteriostat and a deodorant. Previous studies conducted by Lin et al. [12] and Berrang
et al. [4] reported high bactericidal activity of
ClO2 against E. coli, Salmonella, Campylobacter, Bacillus spp., Staphylococcus aureus, or
Sarcina spp. Barreiro et al. [16] supplied chlorinated water to broilers during 12 h of feed
withdrawal before slaughter and reported a significant reduction in the concentration of Enterococci and E. coli in the broiler crop and
ceca. However, no information is available regarding the effects of dietary ClO2 on modulation of the intestinal microbial ecology of broilers. As shown in Table 3, dietary ClO2 had no
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506
Table 2. Effects of dietary chlorine dioxide (ClO2 ) on the growth performance
of broilers.1
Chlorine dioxide (ClO2 )
Performance parameter
0%
0.05%
Body weight (g/bird)
One-day-old
46.13
45.79
D 21
973.06
916.88
D 35
2045.00
1947.50
Average daily gain (ADG, g/bird)
Starter (d 0 to 21)
44.14
41.48
Finisher (d 21 to 35)
76.57
73.62
Overall (d 0 to 35)
57.11
54.34
Average daily feed intake (ADFI, g/bird)
54.96b
Starter (d 0 to 21)
63.52a
135.34b
Finisher (d 21 to 35)
151.37a
87.11b
Overall (d 0 to 35)
98.66a
FCR (feed/gain)
1.33b
Starter (d 0 to 21)
1.44a
1.84b
Finisher (d 21 to 35)
1.98a
1.61b
Overall (d 0 to 35)
1.73a
0.1%
SEM
P-value
45.94
954.38
2019.38
0.48
26.92
34.79
NS
NS
NS
43.26
76.07
56.39
1.27
1.49
0.99
NS
NS
NS
57.61b
138.23b
89.86b
1.64
2.13
1.46
0.0219
0.0023
0.002
1.34b
1.82b
1.59b
0.03
0.03
0.02
0.0471
0.0248
0.0073
a,b
Values with different superscripts in the same row differ significantly (P < 0.05).
Differences between treatment means were separated using a Student’s t test.
1
Each value represents the means of 5 replications with 8 birds/replication.
Table 3. Effects of dietary chlorine dioxide on ileal and cecal microbiology
in broiler chickens.1
Chlorine dioxide (ClO2 )
Microorganisms (log cfu/g)
Ileum
Lactobacillus spp.
Bacillus spp.
Yeast and mold
Escherichia coli
Salmonella Typhimurium
Cecum
Lactobacillus spp.
Bacillus spp.
Yeast and mold
Escherichia coli
Salmonella Typhimurium
0%
0.05%
0.1%
SEM
P-value
5.57
4.63
3.59b
5.29a
0.76
4.59
3.94
5.44a
5.35a
0.25
5.15
3.75
5.63a
4.11b
0.64
0.35
0.27
0.28
0.23
0.38
NS
NS
0.0015
0.011
NS
6.90
6.26a
5.97
6.16a
3.14a
6.89
5.24b
5.43
4.42b
0.34b
6.75
5.24b
6.04
5.00b
0.93b
0.15
0.16
0.30
0.27
0.38
NS
0.0046
NS
0.0065
0.0016
a,b
Values with different superscripts in the same row differ significantly (P < 0.05).
Differences between treatment means were separated using a Student’s t test.
1
Each value represents the mean of 5 replicates (3 birds/replicate).
significant effects on the number of ileal Lactobacillus and Bacillus bacteria (P > 0.05); however, it led to significantly increased numbers of
yeast and mold (P = 0.002). In contrast, ClO2 at
a rate of 0.1% of the diet significantly decreased
the number of ileal E. coli (P < 0.01). There were
no significant differences among dietary treatments for ileal Salmonella concentration. In the
cecal digesta, the concentration of Lactobacillus
bacteria and yeast and mold remained unaffected
in response to dietary ClO2 supplementation, although significant reductions in the number of
Bacillus (P = 0.005), E. coli (P < 0.01), and
Salmonella (P = 0.002) were reported. Dietary
supplementation with ClO2 had no significant
effects on the pH of ileal and cecal digesta (data
not shown).
The effects of dietary ClO2 on excreta microbiota are shown in Table 4. No significant differences were recorded among dietary
AHMED ET AL.: CHLORINE DIOXIDE IN BROILERS
507
Table 4. Effects of dietary chlorine dioxide on fecal microbiology in broiler chickens.
Chlorine dioxide (ClO2 )
Microorganisms (log cfu/g)
Lactobacillus spp.
Bacillus spp.
Yeast and mold
Escherichia coli
Salmonella Typhimurim
Period
0%
0.05%
0.1%
SEM
P-value
21 d
35 d
21 d
35 d
21 d
35 d
21 d
35 d
21 d
35 d
7.47
7.29
7.02
6.56
6.87
6.15
6.59a
5.63a
5.83a
5.51a
7.29
6.97
6.29
6.32
6.58
6.13
5.58b
5.39a
5.02b
4.73a,b
7.37
6.61
6.61
6.61
6.92
6.13
5.60b
4.64b
5.10b
4.07b
0.09
0.23
0.27
0.35
0.18
0.24
0.18
0.20
0.19
0.26
NS
NS
NS
NS
NS
NS
0.0097
0.0162
0.0292
0.0497
Values with different superscripts in the same row differ significantly (P < 0.05).
Differences between treatment means were separated using a Student’s t test.
a,b
Figure 1. Effects of dietary chlorine dioxide (ClO2 ) on fecal pH in broiler chickens at 36 d of age. Treatments at the
same fermentation time not sharing the same letter are significantly different (P < 0.05).
treatments for excreta Lactobacillus, Bacillus,
and yeast and mold concentrations. At d 21, both
levels of ClO2 (0.05 and 0.1%) significantly reduced the number of E. coli (P < 0.01) and
Salmonella (P < 0.03), whereas at d 35, significant reductions were observed only in response
to 0.1% ClO2 (P < 0.05). The primary physiological mode of inactivation of bacteria by ClO2
has been attributed to a disruption in protein synthesis [17]. Specifically, ClO2 exerts direct action on cell membranes by either altering (at high
concentration) or disrupting their permeability
(at low concentration) [9] and then penetrating
into the cell and disrupting the protein synthesis.
However, we did not find any significant effects
of ClO2 on the Gram-positive Lactobacillus and
Bacillus bacteria, except for a significant reduc-
tion in cecum Bacillus content. This may have
occurred because Gram-positive bacteria have
thicker walls than Gram-negative bacteria [18].
A significant increase in the excreta pH was
observed (Figure 1) in response to dietary 0.1%
ClO2 supplementation at 6, 12, and 48 h relative to the non-supplemented group (P < 0.05).
The pH of excreta is an important factor influencing the NH3 emissions from animal manure
[19]. Higher fecal pH favors the conversion of
ammonia-nitrogen to NH3 , allowing for fairly
rapid emission of ammonia into the atmosphere
[20]. In this study, increasing levels of ClO2 led
to increased (P < 0.05) NH3 emissions from
broiler excreta at 48 h (Figure 2), which can
be attributed to the higher fecal pH or the nonreactivity of ClO2 with ammonia-nitrogen [19].
508
JAPR: Research Report
Figure 2. Effects of dietary chlorine dioxide (ClO2 ) on fecal ammonia (NH3 ) emissions at 36 d of age. Treatments
at the same fermentation time not sharing the same letter are significantly different (P < 0.05).
Figure 3. Effects of dietary chlorine dioxide (ClO2 ) on fecal hydrogen sulfide (H2 S) emissions at 36 d of age.
Treatments at the same fermentation time not sharing the same letter are significantly different (P < 0.05).
Figure 4. Effects of dietary chlorine dioxide (ClO2 ) on fecal sulfur dioxide (SO2 ) emissions at 36 d of age. Treatments
at the same fermentation time not sharing the same letter are significantly different (P < 0.05).
The emission rates of excreta sulfuric odorous gases (H2 S, SO2 , and mercaptan) in response
to dietary supplementation of ClO2 are shown in
Figures 3–5, respectively. At 0 h, inclusion of
0.05% ClO2 resulted in a significantly higher
emission of H2 S from broiler excreta relative to
the non-supplemented group (P < 0.05). However, the emission was significantly reduced in
AHMED ET AL.: CHLORINE DIOXIDE IN BROILERS
509
Figure 5. Effects of dietary chlorine dioxide (ClO2 ) on fecal mercaptans emissions at 36 d of age. Treatments at
the same fermentation time not sharing the same letter are significantly different (P < 0.05).
response to 0.1% ClO2 at 3 and 6 h and in response to both 0.05 and 0.1% ClO2 at 12 and
24 h (P < 0.05). In addition, significantly lower
emissions of SO2 were recorded at 24 and 48 h
of incubation when ClO2 was included at the
0.1% level of the basal diet relative to the nonincluded group (P < 0.05). Both levels of ClO2
significantly lowered the mercaptans emissions
from broiler excreta at 0 and 48 h, whereas significantly lower emissions were recorded only
in the 0.1% ClO2 supplemented group at 3 h
relative to the non-supplemented group (P <
0.05). Production of volatile sulfur by anaerobic bacteria involves dissimilatory sulfate reduction and metabolism of sulfur-containing amino
acids [21, 22]. The major sulfur reducing bacteria (SRB) in the gut are Desulfovibrio, Desulfotomaculum [23], E. coli, and Salmonella [24],
which are numerically dominant in feces. In this
study, the reduction of the emission of volatile
sulfur gases (H2 S, SO2 , and mercaptans) from
broiler excreta in response to dietary ClO2 can
be explained by the reduction in the concentration of E. coli and Salmonella together with other
SRB in the gut and excreta of broilers that received the supplemented diet. Studies conducted
by Taylor et al. [25] and Berg et al. [26] also
support the reducing effects of ClO2 on SRB
including E. coli. Chlorine dioxide not only effectively reduces SRB, but also oxidizes the H2 S
they produce.
CONCLUSIONS AND
APPLICATIONS
1. Supplementation of the diet with 0.05 and
0.1% ClO2 can reduce the feed intake and
feed conversion ratio of broilers without affecting the growth rate. In addition, supplementation of broiler diets with ClO2 can
improve the intestinal microbial ecology of
broilers by reducing the concentration of
E. coli (ileum and cecum) and Salmonella
(cecum).
2. There was an effective reduction in the emission of excreta sulfuric odorous gases in response to treatment with ClO2 . The underlying mechanisms of reduced emissions may
be the reduction in the number of fecal sulfate reducing bacteria, including E. coli and
Salmonella.
3. ClO2 is not yet approved as a feed additive
for livestock because higher levels may have
toxic effects. In addition, the increased ammonia emissions together with higher excreta pH reported in this experiment suggest
that further investigations using different
doses are warranted.
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Acknowledgments
We express gratitude to the Tecon. Co. Ltd (project no.
2014–0156) for funding this research and supply of chlorine
dioxide for this experiment.