The effectiveness of peppermint and thyme essential oil mist in reducing
bacterial contamination in broiler houses
D. Witkowska1 and J. Sowińska
Department of Animal and Environmental Hygiene, Faculty of Animal Bioengineering,
University of Warmia and Mazury, Oczapowskiego 5, 10-719 Olsztyn, Poland
ABSTRACT The antimicrobial properties of essential
oils have been demonstrated by various in vitro studies,
whereas their effect on poultry farm hygiene has not
been thoroughly investigated, in particular with refer-
ence to aerial treatment. The present study aims to
assess the antibacterial effects of natural essential oils
in broiler houses. Two experimental rooms were fogged
with aqueous solutions of peppermint and thyme oils.
The control room was sprayed with pure water. The ex-
periment was conducted on broilers aged 1 to 42 d. The
rooms were fogged every 3 d. One day after fogging,
the total counts of mesophilic aerobic bacteria, Entero-
bacteriaceae, and mannitol-positive staphylococci were
determined. Samples were collected from the air, lit-
ter, walls, and drinkers. The results of the study dem-
onstrate that essential oil mist may improve hygiene
Key words: essential oil, bacteria, broiler house, Enterobacteriaceae, Staphylococcus
INTRODUCTION
Broiler production leads to the accumulation of vari-
ous pollutants in poultry houses, including bacteria and
their metabolites: endotoxins, toxic gases, and aroma
compounds (Seedorf et al., 1998; Baykov and Stoya-
nov, 1999; Bakutis et al., 2004; Tymczyna et al., 2007;
Vučemilo et al., 2007; Schulz et al., 2011). Pollution
levels increase steadily, and sick building syndrome is
often reported at the end of the production process.
Microbiological contamination is also observed, in par-
ticular a steep increase in the levels of aerobic meso-
philic bacteria, the predominant group of pathogenic
microbes. The number of bacteria released into the
environment increases as chickens age, and pathogenic
microbes are found as far as 3 km from poultry houses
(Baykov and Stoyanov, 1999).
©2013 Poultry Science Association Inc.
Received February 28, 2013.
Accepted July 1, 2013.
1 Corresponding author: dorota.witkowska@uwm.edu.pl
2834
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
standards in broiler farms. During broiler growth, the
mean total counts of mesophilic bacteria in the rooms
treated with essential oils were lower (P < 0.01 or P <
0.05) in comparison with the control. Enterobacteria-
ceae and staphylococci counts were also higher in the
control group. A single exception was noted in a litter
sample where the mean count of Enterobacteriaceae in
the room fogged with peppermint oil was higher than
in the control. Both oils reduced bacterial counts, but
thyme oil was more effective in reducing coliform bacte-
ria, whereas peppermint oil had a higher inhibitory ef-
fect on the proliferation of staphylococci. These promis-
ing results encourage further research to determine the
optimal doses and the effects of essential oils and their
combinations on the living conditions and health status
of broiler chickens.
2013 Poultry Science 92:2834–2843
http://dx.doi.org/10.3382/ps.2013-03147
In addition to gram-positive cocci (Staphylococcus,
Streptococcus, Micrococcus) and bacilli (Bacillus), poul-
try houses are also colonized by gram-negative bacteria
of the family Enerobacteriaceae, including Escherichia
coli, Salmonella spp., Shigella spp., and Klebsiella spp.,
as well as Pseudomonas spp., Acinetobacter spp., and
Flavobacterium spp. (Davis and Morishita, 2005; Tym-
czyna et al., 2007; Vučemilo et al., 2007; Bródka et
al., 2012). The cell membranes of pathogenic gram-neg-
ative bacteria contain lipopolysaccharide complexes.
Those endotoxins are released when bacterial cells dis-
integrate, and they are present in high concentrations
in poultry houses (Bakutis et al., 2004). Salmonella
bacteria can survive in poultry houses between produc-
tion cycles, including after disinfection, posing a signifi-
cant problem in poultry farms (Wales et al., 2006). The
discussed microbes create a high epidemiological risk,
and restrictive prevention programs, which often lead
to sever losses, have to be implemented. For this rea-
son, alternative solutions are needed to minimize this
problem in poultry farms.
Essential oils have been long known to posses anti-
microbial properties (Mitsch et al., 2004). The results
 EFFECTIVENESS OF ESSENTIAL OIL MIST
of in vitro studies have demonstrated that essential
oils eliminate pathogenic bacteria, among them E. coli,
Salmonella spp. (including Salmonella Enteritidis and
Salmonella Typhimurium), Pseudomonas aeruginosa,
Staphylococcus aureus, Staphylococcus epidermis, Kleb-
siella pneumoniae, Shigella spp., Proteus vulgaris, and
Bacillus cereus (Hammer et al., 1999; Inouye et al.,
2003; Azaz et al., 2004; Bagamboula et al., 2004; Pe-
ñalver et al., 2005; Kędzia and Hołderna-Kędzia, 2007).
Thyme essential oil (Oleum thymi) extracted from
the common thyme (Thymus vulgaris L.) demonstrates
high levels of antimicrobial activity, and it has been
frequently compared with other oils in both in vitro
(Hammer et al., 1999; Lutomski and Kędzia, 2000; Azaz
et al., 2004; Bagamboula et al., 2004; Peñalver et al.,
2005; Al-Bayati, 2008) and in vivo studies (Mitsch et
al., 2004). Thyme plants contain around 0.5 to 2.5% oil,
and thyme essential oil is made up of thymol (45–47%),
p-cymene (32–34%), carvacrol (4–5%), γ-terpinene, lin-
alool, and α-pinene (Bagamboula et al., 2004). The oil
extracted from selected thyme varieties may contain up
to 60% carvacrol (Azaz et al., 2004). Thymol and car-
vacrol (phenolic compounds) are thyme oil ingredients
with the strongest antiseptic properties (Bagamboula
et al., 2004).
Peppermint essential oil (Oleum menthae piperitae),
obtained from peppermint plants (Mentha piperita),
is also a highly potent antimicrobial substance whose
composition differs from thyme oil. Peppermint oil con-
tains mainly menthol (63%) and p-menthone (19.5%)
as well as ketones, monoterpenes, menthofuran, and
terpene oxides (Inouye et al., 2001; Kędzia, 2007).
Essential oils attracted the interest of European an-
imal breeders after the use of antibiotic growth-pro-
moters was banned in animal nutrition. In addition to
herbs, spices, and plant extracts, essential oils offer a
viable alternative to feed antibiotics due to the content
of various active substances. Herbal feed additives are
widely used, and some formulas include essential oils
(Hernández et al., 2004; Mitsch et al., 2004). Commer-
cially available products containing essential oils can be
added to drinking water or used in the fumigation of
poultry houses, but their effects on the living environ-
ment of birds have not been investigated in detail to
date. The use of essential oil vapor could improve poul-
try house hygiene. Inouye et al. (2001, 2003) observed
that essential oils in liquid and vapor form inhibited the
growth of selected bacteria and fungi under laboratory
conditions. The above authors found that essential oil
vapors were much more effective because water vapor
had an additional influence on microbial cells. They
concluded that essential oils should be optimally used
over short periods of time, but at high water vapor con-
centrations. Positive results of in vitro studies encour-
age further research into the effects of essential oils on
the living environment of chickens.
The aim of this study was to evaluate the antibacte-
rial effect of natural thyme and peppermint essential
oils in broiler houses.
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
2835
MATERIALS AND METHODS
Chicken Housing and Experimental Design
The experimental design and procedures were ap-
proved by the Local Ethics Committee for Animal Ex-
perimentation at the University of Warmia and Mazury
in Olsztyn. The experiment was performed in spring on
360 unsexed Ross 308 broiler chickens aged 1 to 42 d.
One-day-old chicks were divided into 3 groups (control
group and 2 experimental groups) of 120 birds each.
Broilers were housed in 3 separate, climate-controlled
(forced-air ventilation), and identically equipped rooms
with a floor area of 9.25 m2 and a cubic capacity of
26.2 m3. Each experimental room was cleaned and dis-
infected before stocking. Chickens were kept on straw
litter in accordance with standard requirements. Initial
ambient temperature was set at 32°C, and it was gradu-
ally decreased to 20°C in wk 6. Each room was provided
with ad libitum access to feed and water. Birds were
fed commercial starter, grower, and finisher pellet diets
(Rolpol, Uścikowo, Poland) containing wheat and soy-
bean extracts and Schaumann premixes (Gniezno, Po-
land). Chicks were vaccinated in the hatchery against
infectious bronchitis, infectious bursal, Marek’s, and
Newcastle diseases.
Commercially available 100% natural essential oils
(ETJA Corp., Elbląg, Poland) were used in the experi-
ment. The experimental broiler rooms were fogged with
aqueous solutions of peppermint oil (Mentha piperita)
and thyme oil (Thymus vulgaris L.). Hot water was
mixed with essential oils in doses recommended by the
manufacturer for human aerosol therapy (based on the
cubic capacity of fogged rooms). The initial concen-
tration of 1:500 was increased to 1:250 at 28 d before
slaughter. The experimental rooms were fogged with 0.5
L of each oil solution before stocking and every third
day during the rearing period. The control room was
sprayed with 0.5 L of pure water. A fogger (Mgła-E,
Poltech Corp., Warsaw, Poland) was used to produce
aerosol particles smaller than 50 μm.
Sampling and Microbiological Analysis
Bacterial contamination of samples collected from the
air, litter, walls, and drinkers was evaluated 1 d after
fogging (every 3 d). Quantitative analyses of mesophil-
ic aerobic bacteria, Enterobacteriaceae pathogens, and
mannitol salt-positive staphylococci were carried out.
Bacterial counts were also determined before stocking
before and after essential oil treatments.
Air samples were collected with an air sampler (air
IDEAL 3P, bioMérieux Corp., Craponne, France) at 5
locations in each room—in the center and at 2 points
along 2 diagonals. The sampler, which operates on the
Andersen impaction principle, has air intake of 100 L/
min and impact speed of less than 20 m/s. Air volumes
ranged between 5 and 100 L, subject to the type of me-
dium and the expected contamination levels. Bacterial
 2836 Witkowska and Sowińska
counts were determined in Petri dishes (90 mm) with
solid media (Merck Corp., Darmstadt, Germany). Me-
sophilic bacteria were cultured on tryptic soy agar with
casein-peptone and soy meal-peptone (TSA). The cul-
tures were incubated at 35°C for 24 h. Enterobacteria-
ceae were cultured on crystal-violet neutral-red bile glu-
cose agar according to Mossel et al. (1962; VRBD) for
24 h at 35°C. Red to dark purple colonies surrounded by
red-purple halos were analyzed. Mannitol and staphylo-
cocci were isolated on mannitol salt agar (MSA), and
the colonies surrounded by a bright yellow zone were
counted after 48 h of incubation at 35°C. The number
of colonies on Petri dishes was determined using an au-
tomated colony counter (Schuett colonyQuant, Schuett-
Biotec Corp., Göttingen, Germany). The results were
corrected using Feller’s conversion formula, and bacte-
rial counts were expressed in terms of cfu per meter3
of air. The microbiological contamination of walls and
drinkers was evaluated based on smear samples col-
lected in accordance with standard PN-ISO 18593:2005
(2005). Two swab samples from the walls and drinkers
in each room were collected in every analytical series.
Sterile templates measuring 10 cm × 10 cm and sterile
cotton swabs moistened with sterile peptone saline were
used to obtain samples from flat wall surfaces. Swab
samples from drinkers were collected from the same
area each time. Swab samples were directly immersed
in peptone saline solution (Merck Corp.) and shaken
vigorously. Bulk samples of litter comprised 5 random
samples collected with a sterile spatula. One gram of
each sample was diluted 1:9 (wt/vol) in sterile peptone
saline. All samples collected from the walls, drinkers,
and litter were subjected to 10 sequential dilutions 1:9
(vol/vol). One milliliter of each dilution was inoculated
in double Petri dishes with TSA, VRBD, and MSA me-
dia (Merck Corp.). The dishes were incubated at 35°C
for 24 h (TSA and VRBD) or 48 h (MSA). Bacterial
colonies were expressed in terms of cfu/100 cm2 of wall
surface, cfu per unit of drinker area, and cfu per 1 g
of litter. Bacterial assays were performed according to
Merck and PN-EN ISO 21528–2:2005 (2005) standards.
Statistical Analysis
Before statistical analysis, all bacterial counts were
log10 transformed to achieve normal distribution and
calculate the mean, SD, minimum, and maximum val-
ues. The data present in Tables 4 to 6 were collected
after fogging (every 3 d) and are reported as an average
of the entire chicken rearing. The normality check for
each group was performed by the Kolmogorov-Smirnov
test. All data were normally distributed and were sub-
jected to one-way ANOVA in Statistica 10 for Win-
dows (StatSoft Inc., Tulsa, OK). The mean values of
treatment groups were compared by Duncan’s multiple
range test, and differences were considered at P < 0.01
and P < 0.05. Figures 1 to 3 show the trend lines of
bacterial reductions by age and essential oil treatment,
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
and the data points are the averages of 2 measurements
from each rearing week.
RESULTS
Tables 1, 2, and 3 present the results of environmen-
tal monitoring of bacteria in control and experimental
rooms before stocking. After the rooms had been pre-
pared for rearing, the average counts of aerial meso-
philic bacteria were similar in each room at around 2.6
log10 cfu/m3. The total number of mesophilic bacteria
in litter was approximately 4.2 log10 cfu/g in 2 rooms,
whereas a difference of 0.3 log10 was reported in the
third room. The contamination of walls was determined
at 1.0 log10/100 cm2, and somewhat higher contamina-
tion levels were noted in drinkers (1.3–1.7 log10). The
number of aerial mannitol-positive staphylococci was
comparable with or lower than total mesophilic counts
(in one of the rooms). Litter was considerably less con-
taminated with staphylococci than with mesophilic
bacteria. On the surfaces of walls and drinkers, staphy-
lococci were not identified or their counts were below
0.7 log10. The presence of Enterobacteriaceae was not
determined in disinfected rooms before stocking with
a single exception in one room where minimal litter
contamination (0.2 log10/g) was observed. Three days
later, 1 d after essential oil fogging, an increase was
reported in mesophilic counts in the air and on control
room surfaces (Table 1). In experimental rooms, the
count of aerial mesophilic bacteria after essential oil
treatment was identical to that noted after disinfection.
The levels of contamination on the surfaces of walls
and drinkers were lower (around 0.6–1.2 log10) in com-
parison with the previous analysis. Mesophilic counts
in litter were similar in each room (around 5.0 log10/g)
and approximately 1.0 log10 higher than during previ-
ous determinations. A similar trend was observed with
regard to staphylococci counts in litter, but in the room
treated with peppermint oil, contamination levels were
roughly 2.0 log10 higher in comparison with the previ-
ous examination. The walls were colonized by staphy-
lococci only in the control room (1.0 log10), whereas
drinkers were contaminated in the control room and
in the room fogged with thyme oil (around 2.0 log10).
In those rooms, staphylococci counts were 2-fold high-
er (above 3.0 log10/m3) in comparison with the room
where peppermint oil was used. Enterobacteriaceae
were identified only in litter, and their population size
ranged from 0.60 (control) to 1.36 (peppermint oil
treatments) log10/g.
The average total mesophilic counts in the air, litter,
and on wall and drinker surfaces during broiler rearing
are presented in Table 4. The mean aerial contamina-
tion of the analyzed rooms ranged from below 5.5 to
above 5.8 log10/m3, and it was higher in the control
room (P = 0.002) than in the rooms treated with es-
sential oils. No statistically confirmed differences were
observed between essential oil groups, but the lowest
 EFFECTIVENESS OF ESSENTIAL OIL MIST 2837

Figure 1. Total mesophilic counts in the air (a), in litter (b), on Figure 2. Enterobacteriaceae counts in the air (a), in litter (b), on
walls (c), and on drinkers (d) from wk 1 to 6. Averages of 2 measure- walls (c), and on drinkers (d) from wk 1 to 6. Averages of 2 measure-
ments each week. ments each week.

Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366

by guest
on 20 November 2017
 2838 Witkowska and Sowińska
Figure 3. Mannitol salt-positive staphylococci counts in the air
(a), in litter (b), on walls (c), and on drinkers (d) from wk 1 to 6.
Averages of 2 measurements each week.
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
mean and maximum values were noted in the room
treated with thyme oil. A similar trend was observed
with regard to wall contamination, but the differences
between control and experimental groups were deter-
mined at P = 0.025 and total mesophilic counts ranged
from around 2.4 in rooms fogged with essential oils to
3.3 log10/100 cm2 in the control room. It should be
noted that the maximum total bacterial concentrations
on control room walls were approximately 5.2 log10,
whereas in rooms fogged with essential oils, the maxi-
mum values did not exceed 3.6 log10 (thyme oil) and
3.8 log10 (peppermint oil). Total bacterial counts on
drinker surfaces in the room treated with thyme oil
were lower (P = 0.045) than in the control room (by
0.5 log10). No statistically confirmed differences were
noted between other groups, but the mean total meso-
philic counts in the room fogged with peppermint oil
were lower in comparison with the control room. The
minimum and maximum values in the room treated
with thyme oil were around 1 logarithm lower than
in the other rooms. The average total count of litter
bacteria was consistently highest in the control group
at approximately 8.9 log10/g, and it was lowest in the
thyme oil group at 8.2 log10/g (P = 0.031). The maxi-
mum and minimum total bacterial counts in litter re-
vealed the same trend (Table 4). Figure 1 presents total
mesophilic counts in the investigated rooms throughout
the entire rearing period. The curve representing total
aerial bacterial populations (Figure 1a) is similar for all
groups, and bacterial counts in each week of the experi-
ment ranged from 4.9 to 5.6 log10/m3 (essential oils) to
5.3 to 6.0 log10/m3 (control), excluding the second week
when they increased to 6.0 (thyme oil), 6.2 (peppermint
oil), and 6.4 log10/m3 (control). Total concentrations
of aerial bacteria were highest in the control room and
lowest in the room treated with thyme oil. A similar
trend was observed on the surface of drinkers and in lit-
ter (Figure 1b and 1d), but total bacterial populations
of walls (Figure 1c) in the second half of the rearing
period were smaller in the room treated with pepper-
mint oil. Total bacterial counts in litter and on drinker
surfaces were characterized by a growing trend in the
control room, whereas greater variations were reported
in experimental rooms. A noticeable drop in the size
of bacterial populations contaminating surfaces was
observed in the third week of the study in the room
treated with thyme oil (Figure 1c).
The mean Enterobacteriaceae counts in birds environ-
ment throughout the entire rearing period are shown in
Table 5. The control room was characterized by the
highest concentrations of aerial bacteria (1.8 log10; P
= 0.041), as well as pathogens colonizing the walls (1.2
log10) and drinkers (2.5 log10). In the experimental
room treated with thyme oil, Enterobacteriaceae counts
on wall surfaces were lower (P = 0.005) than in the
control room (by 1.0 log10). The differences (P = 0.037)
were also observed between the above rooms in the
populations of aerial bacteria and pathogens colonizing
drinkers. Enterobacteriaceae counts determined in the
 EFFECTIVENESS OF ESSENTIAL OIL MIST 2839
Table 1. Total mesophilic counts (log10 cfu) in the air, on walls, on drinkers, and in litter before stocking
Before essential oil treatment After essential oil treatment

Peppermint Peppermint
Place Control oil Thyme oil Control oil Thyme oil

Air (m3) 2.62 2.65 2.59 4.43 2.20 2.64

Walls (100 cm2) 1.23 1.11 0.95 2.41 0.00 0.30
Drinkers (unsized) 1.70 1.45 1.32 3.08 0.30 0.78
Litter (g) 4.22 3.91 4.24 4.89 4.99 5.08

air and on surfaces in the room fogged with pepper-
mint oil were also lower than in the control group, but
the differences were not statistically confirmed. In the
above room, the average coliform counts in litter were
highest (6.4 log10) in comparison with the control room
and the room treated with thyme oil, but the differ-
ences were not statistically confirmed. The maximum
and minimum Enterobacteriaceae concentrations were
consistently lowest in the room where thyme oil was
used. Coliform counts in different weeks of chicken rear-
ing are shown in Figure 2. The curves indicate consid-
erable variations between different weeks and groups.
It should be noted, however, that Enterobacteriaceae
counts in the air (Figure 2a) and on the surfaces of
walls (Figure 2c) and drinkers (Figure 2d) remained
lowest in the room treated with thyme oil throughout
the experiment.
The mean values of mannitol-positive staphylococci
during the experiment are presented in Table 6. The
highest aerial contamination was observed in the con-
trol room (4.8 log10), but contrary to previous results,
the lowest staphylococci counts were determined in the
room treated with peppermint oil. The differences were
observed between the control and the peppermint oil
group (P = 0.003) and between control and the thyme
oil group (P = 0.018). The lowest level of contamina-
tion on wall surfaces (1.2 log10) was also noted in the
room fogged with peppermint oil where staphylococci
counts were 1.0 log10 lower than in control (P = 0.027).
No differences between groups were observed with re-
gard to mean staphylococci populations in litter and
on drinker surfaces. The lowest maximum staphylococ-
ci counts (except litter) were determined in the room
treated with thyme oil. The size of staphylococci popu-
lations in each room in different weeks of the experi-
ment is shown in Figure 3. Similarly to total bacteria
counts, an increase in staphylococci abundance was ob-
served in the second week of the study. In the following
weeks, bacterial levels, in particular concentrations of
aerial pathogens (Figure 3a), were stabilized, but some
variations were noted. In wk 3, a clear decrease was
reported in bacterial concentrations on wall surfaces
(Figure 3c) in the room treated with thyme oil, and it
was accompanied by a decrease in total bacterial counts
(Figure 1c).
DISCUSSION
This study demonstrated that essential oil fogging
improves hygiene standards in broiler houses. The use
of essential oils before stocking reduced bacterial counts
in the air and on wall and drinker surfaces in experi-
mental rooms and reduced the populations of mannitol-
positive staphylococci in the room treated with pep-
permint oil solution. Oil treatment had no significant
effect on litter hygiene (Tables 1 to 3). During broiler
rearing, the mean total counts of mesophilic bacteria,
Enterobacteriaceae, and staphylococci were also high-
est in the control group (Tables 4 to 6). A single ex-
ception was noted in litter where the average Entero-
bacteriaceae concentrations in the room treated with
peppermint oil were higher than in the control. Bac-
terial contamination levels were reduced by both oils,
but thyme oil demonstrated a stronger antibacterial
effect with regard to coliform bacteria. Peppermint oil
proved to be a more potent inhibitor of staphylococci
(Table 6). Hammer et al. (1999) performed an in vi-
tro study investigating the activity of 52 plant oils and
extracts against various bacteria, including Salmonella
Typhimurium, E. coli, Klebsiella pneumoniae, Serratia
marcescens, and Staphylococcus aureus, and thyme oil
was characterized by the lowest minimum inhibitory
concentrations against E. coli. The results of the cited
study demonstrate that thyme oil is a more effective
inhibitor of the above bacteria, excluding Salmonella
Typhimurium, than peppermint oil. Inouye et al. (2001,

Table 2. Enterobacteriaceae counts (log10 cfu) in the air, on walls, on drinkers, and in litter before stocking
Before essential oil treatment After essential oil treatment

Peppermint Peppermint
Place Control oil Thyme oil Control oil Thyme oil

Air (m3) 0.00 0.00 0.00 0.00 0.00 0.00

Walls (100 cm2) 0.00 0.00 0.00 0.00 0.00 0.00
Drinkers (unsized) 0.00 0.00 0.00 0.00 0.00 0.00
Litter (g) 0.00 0.20 0.00 0.60 1.36 1.00

Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366

by guest
on 20 November 2017
 2840 Witkowska and Sowińska
Table 3. Mannitol salt-positive staphylococci counts (log10 cfu) in the air, on walls, on drinkers, and in litter before stocking
Before essential oil treatment
Peppermint
Place Control oil
Air (m3) 2.03 2.28
Walls (100 cm2) 0.00 0.30
Drinkers (unsized) 0.73 0.00
Litter (g) 1.57 1.10
2003) also observed that thyme oil is one of the most
effective agents against respiratory tract pathogens by
gaseous contact in laboratory conditions. In the above
study, thyme oil had greater antibacterial activity
against E. coli, S. aureus, and other bacteria than pep-
permint and other oils. The antimicrobial properties of
thyme and other essential oils have been demonstrat-
ed by various in vitro studies (Friedman et al., 2002;
Azaz et al., 2004; Bagamboula et al., 2004; Al-Bayati,
2008). Peñalver et al. (2005) analyzed the effect of es-
sential oils on pathogenic intestinal Enterobacteriaceae
strains of poultry origin. They reported the most sat-
isfactory results in respect of oils containing carvacrol
and thyme. Their findings justify further research into
the use of essential oils for the prevention of diseases
caused by Salmonella. In another study, the addition of
essential oils to feed significantly lowered Clostridium
perfringens counts in the intestines and feces of broil-
ers (Mitsch et al., 2004), thus lowering the incidence of
necrotic enteritis (Jerzsele et al., 2012). Diets supple-
mented with plant extracts and essential oils improved
nutrient digestibility and performance results in broiler
chickens (Hernández et al., 2004; Cross et al., 2007).
According to Mickienė et al. (2011), selected essential
oils offer a promising alternative to other air disinfec-
tion methods in animal houses. The effect of essential
oils on hygiene standards during broiler production has
not been thoroughly investigated. Mituniewicz et al.
(2008) applied Profistreu, a commercial preparation
containing organic and inorganic active substances as
well as essential oil to litter, and observed an average
reduction of 0.33 log10 in total counts of aerial bacte-
ria in broiler houses. Bakutis et al. (2011) investigated
the effectiveness of pine and eucalyptus oils and their
combinations in a hen house. A combination of the ana-
lyzed oils proved to be the most effective inhibitor of
aerial bacteria in the above experiment. In our investi-
gation, a decrease in total aerial bacterial counts was
also noted, but our experiment differed with regard to
the applied types and concentrations of essential oil,
the form and period of their administration, the evalu-
ated species, and environmental conditions. In a study
by Vučemilo et al. (2007), microbial pollution levels in
poultry houses varied significantly due to differences in
housing standards and the applied research methods.
In a 6-wk-long study of a commercial farm, Vučemilo
et al. (2007) reported an increase in bacterial counts
from 4.2 log10 cfu/m3 in the first week to 5.3 log10 cfu/
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
After essential oil treatment
Peppermint
Thyme oil Control oil Thyme oil
2.62 3.19 1.54 3.31
0.00 1.00 0.00 0.00
0.47 1.76 0.00 2.00
2.25 3.30 3.64 2.96
m3 in the fifth week. In an experiment by Baykov and
Stoyanov (1999), bacterial counts increased from 3.1
log10 cfu/m3 at the beginning of the production cycle
to 5.2 log10 cfu/m3 on d 56. In our study, a noticeable
increase in the counts of aerial bacteria was reported in
the second week of broiler rearing (Figure 1). Similar
results were reported by Wójcik et al. (2010) who ob-
served differences in bacterial concentrations between
winter and summer seasons.
In a study by Brooks et al. (2010), staphylococci ac-
counted for at least 90% of cultured aerobic bacteria in
broiler houses. According to Schulz et al. (2011), staph-
ylococci are more useful indicators of bioaerosol emis-
sions from broiler houses than total bacterial counts
that may also represent other sources of pollution. In
our study, aerial staphylococci were characterized by
a similar proliferation pattern to that of total bacte-
ria during broiler rearing (Figure 3a). The reduction in
Staphylococcus populations in the rooms treated with
essential oils was similar to the decrease in total cell
counts.
Essential oils were most effective in eliminating En-
terobacteriaceae colonizing wall surfaces whose popula-
tions were reduced by 80% in the room treated with
thyme oil. Kašková et al. (2006) investigated the ef-
fectiveness of various sanitation methods in poultry
houses to observe that mechanical cleaning decreased
Enterobacteriaceae counts on wall surfaces by 86%. In
general, surface cleaning and disinfection methods ap-
plied in the above experiment were more effective in
eliminating pathogens than the essential oils investi-
gated in our study.
Essential oils were the least potent inhibitors of mi-
crobial activity in litter. Pope and Cherry (2000) ob-
served that PLT (Poultry Litter Treatment, composed
of sodium bisulfate) eliminated litter bacteria, but the
noted results were not statistically confirmed. They
concluded that PLT has the potential to reduce E. coli
and Salmonella populations in broiler house litter, but
it is not capable of eliminating those pathogens entirely.
Our results indicate that essential oil fogging in
poultry houses improves hygiene standards, but their
efficacy seems to be lower than that of conventional
disinfectants used by the authors mentioned previously
in the Discussion. Therefore, there is a need to com-
pare their effectiveness to the efficacy of disinfectants.
The chemical composition of essential oils is difficult to
standardize (Peñalver et al., 2005; Cross et al., 2007).
by guest
on 20 November 2017
Table 4. Total mesophilic counts (log10 cfu) in the air, on walls, on drinkers, and in litter during broiler rearing1
Control Peppermint oil Thyme oil

Place n P-value Mean Minimum Maximum SD Mean Minimum Maximum SD Mean Minimum Maximum SD

Air(m3) 60 0.002 5.81A 4.75 6.66 0.42 5.61B 4.56 6.44 0.49 5.46B 4.59 6.37 0.46
Walls (100 cm2) 24 0.025 3.27a 1.45 5.19 1.05 2.36b 0.60 3.80 0.98 2.43b 1.00 3.64 1.02
Drinkers (unsized) 24 0.045 4.81a 3.20 5.64 0.73 4.63ab 3.43 5.46 0.56 4.30b 2.22 4.93 0.79
Litter (g) 24 0.031 8.92a 7.53 9.86 0.68 8.45ab 7.18 9.52 0.70 8.20b 7.00 9.22 0.81
A,BMeans within a row with different superscripts differ (P < 0.01).
a,bMeanswithin a row with different superscripts differ (P < 0.05).
1Mean values are the averages of each measurement (every 3 d) from wk 1 to 6.

Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366

Table 5. Enterobacteriaceae counts (log10 cfu) in the air, on walls, on drinkers, and in litter during broiler rearing1
Control Peppermint oil Thyme oil
EFFECTIVENESS OF ESSENTIAL OIL MIST

Place n P-value Mean Minimum Maximum SD Mean Minimum Maximum SD Mean Minimum Maximum SD

Air(m3) 60 0.041 1.79a 0.00 3.16 0.78 1.61ab 0.00 2.96 0.90 1.47b 0.00 2.78 0.81
Walls (100 cm2) 24 0.005 1.21A 0.00 2.72 1.02 0.81AB 0.00 2.54 0.61 0.24B 0.00 1.04 0.21
Drinkers (unsized) 24 0.037 2.45a 1.00 3.45 0.69 2.34ab 0.85 3.92 0.90 1.67b 0.30 2.97 0.91
Litter (g) 24 0.488 6.28 4.48 8.15 1.08 6.42 5.00 8.08 1.06 6.09 4.48 8.08 1.22
A,BMeans within a row with different superscripts differ (P < 0.01).
a,bMeans within a row with different superscripts differ (P < 0.05).
1Mean values are the averages of each measurement (every 3 d) from wk 1 to 6.
2841
 2842 Witkowska and Sowińska

The main advantage of essential oils is that they do not

0.54
1.07
1.35
1.47
lead to the increase of microbial resistance, and unlike

SD
disinfectants and antibiotics, oil residues are not found
in final products (Peñalver et al., 2005). The results of

Maximum
our study encourage further research to determine the

5.36
2.48
4.23
9.40
optimal doses and effects of essential oils and their com-
Thyme oil
binations on the living environment and health status
of broiler chickens.
Minimum

2.99
0.00
0.00
4.48
ACKNOWLEDGMENTS
The authors thank Edyta Mituniewicz and Jerzy
1.50ab
Mean

4.54b

Powązka (Faculty of Animal Bioengineering, University

3.30
7.30

of Warmia and Mazury, Olsztyn, Poland) for their tech-

nical support.
Table 6. Mannitol salt-positive staphylococci counts (log10 cfu) in the air, on walls, on drinkers, and in litter during broiler rearing1

1.24
1.12
1.38
0.92
SD

REFERENCES
Maximum

Al-Bayati, F. A. 2008. Synergistic antibacterial activity between

5.94
2.90
6.02
8.26

Thymus vulgaris and Pimpinella anisum essential oils and metha-

nol extracts. J. Ethnopharmacol. 116:403–406.
Peppermint oil

Azaz, A. D., H. A. Irtem, M. Kurkcuoğlu, and K. H. C. Baser. 2004.

Composition and the in vitro antimicrobial activities of the es-
sential oils of some Thymus species. Z. Naturforsch. C. J. Biosci.
Minimum

59:75–80.
1.78
0.00
1.00
5.08

Bagamboula, C. F., M. Uyttendaele, and J. Debevere. 2004. Inhibi-

tory effect of thyme and basil essential oils, carvacrol, thymol,
estragol, linalool and p-cymene towards Shigella sonnei and S.
flexneri. Food Microbiol. 21:33–42.
4.47B,b
Mean

Bakutis, B., V. Baliukoniene, and R. Mickienė. 2011. The use of es-

1.20b
3.65
7.15

sential oils to improve of environment quality in poultry houses.

Proc. 15th ISAH Congress, Vienna, Austria. 2:643–645.
Bakutis, B., E. Monstviliene, and G. Januskeviciene. 2004. Analyses
of airborne contamination with bacteria, endotoxins and dust in
livestock barns and poultry houses. Acta Vet. (Brno) 73:283–
0.74
0.94
1.38
1.27
SD

289.
Baykov, B., and M. Stoyanov. 1999. Microbial air pollution caused
by intensive broiler chicken breeding. FEMS Microbiol. Ecol.
Maximum

1Mean values are the averages of each measurement (every 3 d) from wk 1 to 6.

29:389–392.
6.05
3.18
5.00
8.86

Bródka, K., A. Kozajda, A. Buczyńska, and I. Szadkowska-Stańczyk.

2012. The variability of bacterial aerosol in poultry houses de-
pending on selected factors. Int. J. Occup. Med. Environ. Health
Control

25:281–293.
within a row with different superscripts differ (P < 0.01).
within a row with different superscripts differ (P < 0.05).
Minimum

Brooks, J. P., M. R. McLaughlin, B. Scheffler, and D. M. Miles.

3.05
0.00
0.00
5.08

2010. Microbial and antibiotic resistant constituents associated

with biological aerosols and poultry litter within a commercial
poultry house. Sci. Total Environ. 408:4770–4777.
Cross, D. E., R. M. McDevitt, K. Hillman, and T. Acamovic. 2007.
The effect of herbs and their associated essential oils on perfor-
4.79A,a
Mean

2.22a

mance, dietary digestibility and gut microflora in chickens from 7

3.65
7.73

to 28 days of age. Br. Poult. Sci. 48:496–506.

Davis, M., and T. Y. Morishita. 2005. Relative ammonia concentra-
tions, dust concentrations, and presence of Salmonella species
P-value

and Escherichia coli inside and outside commercial layer facili-

0.003
0.027
0.353
0.092

ties. Avian Dis. 49:30–35.

Friedman, M., P. R. Henika, and R. E. Mandrell. 2002. Bactericidal
activities of plant essential oils and some of their isolated con-
stituents against Campylobacter jejuni, Escherichia coli, Listeria
60
24
24
24
n

monocytogenes, and Salmonella enterica. J. Food Prot. 65:1545–

1560.
Hammer, K. A., C. F. Carson, and T. V. Riley. 1999. Antimicrobial
activity of essential oils and other plant extracts. J. Appl. Mi-
Drinkers (unsized)

crobiol. 86:985–990.
Walls (100 cm2)

Hernández, F., J. Madrid, V. García, J. Orengo, and M. D. Megías.

A,BMeans

2004. Influence of two plant extracts on broilers performance,

a,bMeans
Litter (g)

digestibility, and digestive organ size. Poult. Sci. 83:169–174.

(m3)

Inouye, S., S. Abe, H. Yamaguchi, and M. Asakura. 2003. Compara-

Place

tive study of antimicrobial and cytotoxic effects of selected es-

Air

Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366

by guest
on 20 November 2017
 EFFECTIVENESS OF ESSENTIAL OIL MIST
sential oils by gaseous and solution contacts. Int. J. Aromather.
13:33–41.
Inouye, S., T. Takizawa, and H. Yamaguchi. 2001. Antibacterial ac-
tivity of essential oils and their major constituents against re-
spiratory tract pathogens by gaseous contact. J. Antimicrob.
Chemother. 47:565–573.
Jerzsele, A., K. Szeker, R. Csizinszky, E. Gere, C. Jakab, J. J. Mallo,
and P. Galfi. 2012. Efficacy of protected sodium butyrate, a pro-
tected blend of essentials oils, their combination, and Bacillus
amyloliquefaciens spore suspension against artificially induced
necrotic enteritis in broilers. Poult. Sci. 91:837–843.
Kašková, A., O. Ondrašovičová, J. Sokol, M. Vargová, S. Šmirjaková,
M. Ondrašovič, and M. Chovanec. 2006. Efficiency of sanitary
treatment in poultry breeding and poultry meat processing plant.
Acta Vet. (Brno) 75:611–617.
Kędzia, A. 2007. The activity of peppermint oil (Oleum menthae
piperitae) to anaerobic bacteria. Post. Fitoter. 4:182–186. (in
Polish).
Kędzia, B., and E. Hołderna-Kędzia. 2007. Studies on effect of vola-
tile oils on pathogenic bacteria, yeast fungi and dermatophytes.
Post. Fitoter. 2:71–77. (in Polish).
Lutomski, J., and B. Kędzia. 2000. The estimation of volatile oils
and their compounds in respect to the antiinflammatory and im-
munostimulatory effects. Post. Fitoter. 1:32–35. (in Polish).
Mickienė, R., B. Bakutis, and V. Baliukonienė. 2011. Antimicro-
bial activity of two essential oils. Ann. Agric. Environ. Med.
18:139–144.
Mitsch, P., K. Zitterl-Eglseer, B. Köhler, C. Gabler, R. Losa, and I.
Zimpernik. 2004. The effect of two different blends of essential oil
components on the proliferation of Clostridium perfringens in the
intestines of broiler chickens. Poult. Sci. 83:669–675.
Mituniewicz, T., J. Sowińska, A. Wójcik, K. Iwańczuk-Czernik, D.
Witkowska, and J. Banaś. 2008. Effect of disinfectants on physi-
cochemical parameters of litter, microbiological quality of poul-
try house air, health status and performance of broiler chickens.
Pol. J. Environ. Stud. 17:745–750.
Mossel, D. A. A., W. H. J. Mengerink, and H. H. A. Scholts. 1962.
Use a modified MacConkey agar medium for the selective growth
and enumeration of all Enterobacteriaceae. J. Bact. 84:381–383.
Downloaded from https://academic.oup.com/ps/article-abstract/92/11/2834/1602366
by guest
on 20 November 2017
2843
Peñalver, P., B. Huerta, C. Borge, R. Astorga, R. Romero, and A.
Perea. 2005. Antimicrobial activity of five essential oils against
origin strains of the Enterobacteriaceae family. APMIS 113:1–6.
PN-ISO 18593:2005. 2005. Food and animal feeding stuffs micro-
biology. Horizontal methods of sampling from the surface using
contact plates and swabs. Polish Committee for Standardization,
Warsaw, Poland (in Polish).
PN-ISO 21528–2:2005. 2005. Food and animal feeding stuffs micro-
biology. Horizontal method of Enterobacteriaceae detection and
enumeration—Part 2: Plate method. Polish Committee for Stan-
dardization, Warsaw, Poland (in Polish).
Pope, M. J., and T. E. Cherry. 2000. An evaluation of the presence
of pathogens on broilers raised on Poultry Litter Treatment®-
treated litter. Poult. Sci. 79:1351–1355.
Schulz, J., L. Formosa, J. Seedorf, and J. Hartung. 2011. Measure-
ment of culturable airborne staphylococci downwind from a natu-
rally ventilated broiler house. Aerobiologia 27:311–318.
Seedorf, J., J. Hartung, M. Schröder, K. H. Linkert, V. R. Phillips,
M. R. Holden, R. W. Sneath, J. L. Short, R. P. White, S. Ped-
ersen, H. Takai, J. O. Johnsen, J. H. M. Metz, P. W. G. Groot
Koerkamp, G. H. Uenk, and C. M. Wathes. 1998. Concentra-
tions and emissions of airborne endotoxins and microorganisms
in livestock buildings in Northern Europe. J. Agric. Eng. Res.
70:97–109.
Tymczyna, L., A. Chmielowiec-Korzeniowska, and A. Drabik. 2007.
The effectiveness of various biofiltration substrates in removing
bacteria, endotoxins and dust from ventilation system exhaust
from a chicken hatchery. Poult. Sci. 86:2095–2100.
Vučemilo, M., K. Matković, B. Vinković, S. Jakšić, K. Granić, and
N. Mas. 2007. The effect of animal age on air pollutant concen-
tration in a broiler house. Czech J. Anim. Sci. 52:170–174.
Wales, A., M. Breslin, and R. Davies. 2006. Assessment of cleaning
and disinfection in Salmonella-contaminated poultry layer houses
using qualitative and semi-quantitative culture techniques. Vet.
Microbiol. 116:283–293.
Wójcik, A., Ł. Chorąży, T. Mituniewicz, D. Witkowska, K. Iwańczuk-
Czernik, and J. Sowińska. 2010. Microbial air contamination in
poultry houses in the summer and winter. Pol. J. Environ. Stud.
19:1045–1050.