INTRODUCTION
In the world production of poultry, the share of
waterfowl meat is 7.2%, specifically duck meat – 4.2%,
goose meat – 3%. Their share in the gross production
of poultry meat tends to increase. In industrial poultry
farming, the problem of controlling bacterial infections
of waterfowl is of genuine concern. Salmonella and
Campylobacter are considered the most common
etiological zoonotic factors worldwide, with productive
poultry being the main source of infection.
In recent years, there has been an increase in the
relative number of infections caused by Salmonella
spp. and Campylobacter spp. The microorganisms are
widespread in most warm-blooded and farm animals,
including poultry. Ducks’ infection with salmonella can
be detected at the age of about 14 days, and by the end
of cultivation the whole flock can be found infected.
Experimental studies showed that a small dose (less than
40 CFU) of S. enteritidis is sufficient to fully colonize
the poultry intestines. This can lead to complete flock’s infection in 48 h [5–7]. Microorganisms can colonize
the intestinal tract of poultry in large quantities, often at
above 106–108 CFU/g of intestinal contents. The highest
concentrations of bacterial pathogens are known to be
present in the intestinal mucosa [4].
Poultry products can be contaminated at many
stages of the “from farm to table” food chain, but the
strategic one is the stage of primary poultry production.
Following biosafety guidelines of GMP/HACCP
significantly reduces the colonization of poultry by
bacterial pathogens and, later, the contamination of
carcasses during processing. The European Food Safety
Agency’s monitoring (2008–2018) showed that about
86% of poultry carcasses in Europe were contaminated
with Campylobacter and Salmonella bacteria.
In poultry production, main methods of infection
control are taken at the stage of the cultivation in farms.
Environmentally safe methods that ensure poultry
quality and safety hold promise. Effective systems
of poultry cultivation, feeding, and maintenance
are reqired to control the spread of Salmonella and
Campylobacter in poultry products. Bio-safety
measures, decontamination of dropping and water are
potentilly productive. Antibacterial drugs in treating
of bacterial infections in poultry are considered a risk
factor contributing to the development of antibioticresistant
strains.
Following the trend of avoiding antibiotics, the
search for new control methods is becoming increasingly
important in poultry farming. The application
of antimicrobial alternatives is highly potential.
They include feed additives that are inhibitors of
bacterial pathogens, as well as probiotics, prebiotics,
bacteriophages, bacteriocins, which in combination
prevent antibiotic-resistant strains of microorganisms
and inhibit their proliferation [9–12].
Consequently, natural alternative antibacterial
preparations are a way to reduce poultry gut
colonization by pathogenic microflora. This is the
most acceptable natural alternative to salmonella and
campylobacter control that is economically viable and
does not pose a risk to human health, animals, or the
environment [3, 9]. Effective protection of poultry
against pathogens, naturalness and safety, growth
promotion, and economic effectiveness are the criteria
for new alternatives to antibiotics [11, 13].
One of the requirements for probiotics use is the
competitiveness of antagonistic microflora found in
them. In order to prevent intestinal colonization by
bacterial pathogens, probiotics are recommended for use
from the first day of the birds’ life. Prebiotics promote
the development of birds’ own symbiotic microflora,
which can inhibit pathogens and reduce their adhesion to
enterocytes.
Research suggests that some natural compounds
have biological activity against salmonella proliferation,
but few have shown efficacy in experiments on animals.
“Actigen” prebiotic (Alltech) is a concentrated pure
fraction of mannan oligosaccharides isolated from the
cell walls of Saccharomyces cerevisiae yeast. The main
advantage of these complex carbohydrates is their ability
to adsorb certain strains of bacteria that have type I
fimbriae (mannose-sensitive) and prevent intestinal
colonization by pathogens. Besides, the industrial
experiment proved the influence of combined use of
mannan oligosaccharides and probiotics on intestinal
microbiocenosis and duck productivity [14, 15].
We aimed to develop a method for preventing
bacterial infections and increasing duck productivity
using probiotics and prebiotics. The method was
based on the study of adsorbing capacity of mannan
oligosaccharides (MOS) and antagonistic properties
of Bifidobacterium spp. and Lactobacillus spp. against
Salmonella enteritidis and Campylobacter jejuni. We
also aimed to analyze a combined effect of the cultures
on gut microbiocenosis (activity and colonization) and
on productivity of ducks.
STUDY OBJECTS AND METHODS
We used Salmonella enteritidis and Campylobacter
jejuni isolated from poultry carcasses of Ukrainian
farms. The studies were carried out in 2014–2018 at
Sumy National Agrarian University, Sumy. The poultry
carcasses were subjected to a detailed examination
for pathomorphological changes. The liver, muscles,
cloaca contents, ovaries, and various segments of the
ovoid were aseptically assembled to be screened for
salmonellosis and campylobacteriosis. Isolation and
identification of microorganisms was carried out using
tests recommended by “Bergey’s Manual” (1997) [35].
At the first stage (in vitro), we studied the ability of
mannan oligosaccharides isolated from the cell walls
of Saccharomyces cerevisiae yeast to adsorb bacterial
pathogens. In our experiments we used 27 strains of
Salmonella enteritidis and 13 strains of Campylobacter
jejuni isolated from ducks’ chilled carcasses (liver,
muscles, cloaca).
We used the daily agar culture of bacteria with 1% red
blood cells of guinea pigs. Salmonella (1.5×109 CFU/mL)
was used as an antigen. Erythrocytes were derived
from the blood of a pre-selected donor (guinea pigs).
Blood was placed in flasks containing sodium citrate
and filtered through a cotton gauze filter to remove
fibrin and small blood clots. Blood was centrifuged
with sodium chloride isotonic solution four times
(1500 rpm, 10 min). Then we introduced it into a 10%
suspension of phosphate buffer solution (pH 7.0–7.2).
The washed red blood cells were stabilized with
0.2% acrolein (acrylic aldehyde) solution in the
phosphate buffer (1:1) and incubated in water bath
at 37°C for 30–40 min while stirring periodically.
Erythrocytes were washed three times by centrifuging
with phosphate buffer at 5000 rpm. To improve the
sorption properties of red blood cells, we treated
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them with tannin, combining equal parts 5% of
frozen stabilized red blood cells and tannin solution
(1:30 000). The mixture was left in the thermostat
at 37°C for 40 min, then it was washed twice with
phosphate buffer solution (pH 7.2–7.4) and then twice
with sodium chloride isotonic solution (pH 7.2–7.4).
To sensitize the antigen, we used a 1% red blood cell
suspension. Suspensions were left for 24 h at 4°C to
exclude spontaneous hemagglutination.
The degree of agglutination of the salmonellas
isolated was determined by combining prepared
suspended microorganisms and the aqueous solution
of mannan oligosaccharides (0.2, 0.3 and 0.4 g/L) in a
ratio of 1:1. E. coli O2 test culture was used as a positive
control of the agglutination level of the pathogen.
One-percent red blood cell suspension in phosphate
buffer solution (pH 7.2–7.4) was used as a negative
control [16–18].
At the second stage, we studied the influence
of fraction (Aktigen, Alltech Inc.) on the activity,
colonization and species composition of the microflora
of young ducks’ intestines. Sixty male ducklings
aged 30 days were used in the study. Each experiment
involved one control and two experimental groups
(50 heads in each). First experimental group was
infected with Salmonella enteritidis, and the other
group with Campylobacter jejuni (1×104 CFU/mL
per os). Ducklings were kept in sterile boxes on the
floor and fed by standards. They had free access to feed
and water. In experimental groups, the birds received
a prebiotic fraction of MOS (0.4 kg/t) together with the
feed. Ten days after the infection we determined the
concentration of salmonellas, campylobacil, lactobacil,
bifidobacterium, and total concentration of anaerobic
bacteria using dilution plate counting.
At the third stage, we determined the antagonistic
activity of Bifidobacterium spp. (1.0×109 CFU/mL):
Bifidobacterium lactis, Bifidobacterium longum,
Bifidobacterium bifidum and Lactobacillus spp.
(1.0×109 CFU/mL): (Lactobacillus fermentun, Lactobacillus
salivarius, Lactobacillus acidophilus against
Salmonella enteritidis and Campylobacter jejuni
isolates. Suspensions of bacterial probiotic cultures
in a concentration of 1×109 m.c/cm3 were sown on
Petri dishes and incubated for 24 h at 37°C. After
that, suspensions with microorganisms (Salmonella
enteritidis and Campylobacter jejuni) in a concentration
of 1×109 m.c/cm3 were inoculated by streaking. The
dishes with inoculation were incubated at 37°C for
24–72 h. We recorded the diameter of zones with no
growth of test cultures. To control microbial growth,
we used Preston-agar for Campylobacter, “Salmonella
different agar” for Salmonella, as well as MPA and
MPB for probiotics.
We used the Star 53 H.Y. cross ducklings to
determine the effectiveness of probiotics, prebiotics,
and their combination. The birds were randomly divided
into 4 groups, 123 birds in each. Each group included 3
flocks, 41 birds in each (12 flocks in total). The control
group received the main diet only. Three experimental
groups received three different supplements in addition
to the main diet: bifidobacteria (1.5×109 CFU/mL),
mannan oligosaccharides (“Actigen” prebiotic), and a
combination of Bifidobacterium spp. and Lactobacillus
spp. (1.5×109 CFU/mL) in a ratio of 1:1 and the fractions
of mannan oligosaccharides (0.4 kg/t of feed). These
supplements were mannan-rich fractions isolated from
Table 1 Diet composition
Ingredients Starter Grower
Wheat 55.00 62.00
Full fat whole soya 12.00 12.00
Soybean neal 23.00 20.00
Limestone 0.72 0.50
Di-calcium phosphate 1.65 1.85
Soybean oil 4.50 5.00
Salt 0.20 0.20
Sodium bi-carbonate 0.18 0.16
DL Methionine 0.50 0.40
L-Lysine 0.37 0.30
Threonine 0.25 0.13
Vitamin-mineral premix 0.50 0.50
Nutrient analysis, %, or as indicated
Metabolic Energy, kcal/kg 3000 3125
Crude Protein 24.10 22.00
Lysine 1.42 1.35
Methionine+Cysteine 1.10 0.93
Calcium 1.05 0.85
AVAILABLE PHOsphorous 0.50 0.42
Vitamin-Mineral Premix1
Copper, mg 15.00 15.00
Iodine, mg 1.00 1.00
Iron, mg 30.00 30.00
Manganese, mg 112.00 112.00
Selenium, mg 0.40 0.40
Zinc, mg 105 105
Synergen2, g 158 158
Vitamin A (IU) 13.00 12.00
Vitamin D3 (IU) 4.75 4.50
Vitamin E (IU) 70.00 50.00
Vitamin K, mg 3.00 2.75
Thiamin (B1), mg 3.00 2.50
Riboflavin (B2), mg 10.00 8.00
Niacin, mg 55.00 50.00
Pantothenic Acid, mg 17.00 15.00
Pyridoxine (B6), mg 5.00 4.50
Biotin, mg 0.30 0.25
Folic Acid, mg 2.00 1.70
Vitamin B12, mg 200.00 185.00
Vitamin C, mg 200.00 200.00
Choline, mg 475.00 450.00
1Vitamin-Mineral Premix manufactured by Target Feeds, Shropshire,
UK
2Synergen (g) is a commercial enzyme product by Alltech, Inc.
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Figure 1 Research scheme
the cell wall of Saccharomyces cerevisae yeast. The
main diets were prepared at a commercial feed mill and
consisted mainly of wheat and soybean flour, as shown
in Table 1 [19, 20].
The birds were given starter diets from hatch till day
20, grower diets – from day 21 to 49. Feed and water was
provided throughout the whole study period. Initially,
the room temperature was maintained at 30°C for
10 days, and then gradually decreased every second day
by 1°C. During the experiment, the lighting regime was
the following: for 16 h – light, 8 h – darkness, which
lasted 49 days. All conditions were the same for all the
four groups. The birds were weighed when hatched,
on days 21 and 49. We also measured feed intake to
ISOLATING OF SALMONELLA SPP. AND CAMPYLOBACTER SPP.
FROM REFRIGERATED CARCASSES OF DUCKS (BS EN ISO 10272:2006)
Stage I (In vitro)
Studying of Salmonella absorption by mannan-rich fraction isolated
from cell walls of Saccharomyces cerevisiae yeast
Susceptibility of Bifidobacterium spp.
and Lactobacillus spp. to Salmonella and Campylobacter
Stage II (In vivo)
Studying of the effect of mannan-rich fractions on the activity, colonization
and microflora composition in the gastrointestinal tract of ducks
Infecting ducks with Salmonella enteritidis
and Campylobacter jejuni (1× 104CFU/ml)
Experimental group
received mannan-rich fractions
with a feed (0.4 kg/t)
Control group received
feed (base diets)
Studying of effect of mannan-rich fractions on the concentration
of the intestinal microflora in the ducks’ cecum, log CFU/g
Studying of effect of mannan-rich fractions on the reisolation
of S. enteritidis and C. jejuni from infected poultry
Stage III
Control group
(base diet)
Experimental group (base
diet with probiotics)
Experimental group (base diet
with mannan-rich fractions)
Experimental group (base
diet with probiotics and
mannan-rich fractions)
STUDYING OF EXPERIMENTAL DIETS
ON DUCKS’ GROWTH RATE
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estimate feed conversion rates and body weight gains.
The intact parts of the cecum were withdrawn from 10
randomly caught birds aged 49 days immediately after
euthanasia. The contents of the cecum were placed in
sterile test tubes. Then, the tubes were instantly frozen
in liquid nitrogen, lyophilized and stored at – 80°C for
further analysis.
No principles of the bioethics code were violated
during the experiments [21].
The general scheme of our experimental and
practical studies is shown in Fig. 1.
Bacteriological analysis. We tested the laying houses
for Campylobacter spp. before placing the birds there
and on day 21. According to the methods described in
BS EN ISO 10272:2006, the swabs were placed in 50 mL
of isotonic solution and kept at 260 rpm for one minute
[23, 24]. The suspension (0.1 cm3) was then transferred
to two dishes with breeding ground (Preston agar base
for Campylobacter) and incubated in microaerobic
atmosphere (85% N2, 10% CO2 and 5% O2) at 40.5 ± 1°C.
Then we examined them in 44 ± 4 h for typical and/or
suspicious Campylobacter spp colonies.
Then, Salmonella enteritidis was isolated from the
material. The serotyping of Salmonella spp. was carried
out according to the methods with some modification
according to the data [28, 32–34].
Statistical analysis. Weight gains and feed
conversion rates were studied for statistical group
differences using the Student’s T-test. The results of the
microbiological analysis were logarithmic and evaluated
for the statistical difference between the indicators that
were measurable.
RESULTS AND DISCUSSION
The aim of our research was to study effects of
mannan oligosaccharides fractions and probiotics on
Salmonella enteritiadis and Campylobacter jejuni.
In vitro experiments showed that 0.2–0.4% aquatic
fractions of mannan oligosaccharides could adsorb
all the Salmonella strains and E. coli O 2 test cultures
(positive control).
We detected the most active and pronounced ability
to adsorb bacterial pathogens in in vitro experiments
with 0.4% aqueous fraction of mannan oligosaccharides.
We recorded the beginning of the adsorption process
within 2 min. The active process was manifested in
the form of finely-divided sediment and clearing of
the supernatant. In 8–10 min we observed significant
sedimentation (Fig. 2 a–d).
The formation of the sediment illustrates the
adsorption process that occurred in the test tube.
The same process can occur in the gut in animals and
poultry.
Intestinal colonization by pathogens begins with
the binding of cells to the epithelium of the intestinal
mucosa [17]. Pathogens, including most types of
Salmonella, E. coli, and Campylobacter attach to the gut
via receptors (fimbriae) specific to certain carbohydrates
containing mannose, which localize on the surface of
intestinal mucosal epithelium cells [14].
When entering the intestines of poultry with feed,
mannan-rich fractions bind to receptors of bacterial cells
that have type I fimbriae (mannose-sensitive). Fractions
of mannan oligosaccharides are not broken down by
digestive enzymes and are held firmly on the surface of
bacteria. Bacteria with blocked receptors cannot gain a
foothold on the surface of epithelial cells – they transit
through the gastrointestinal tract [13]. Thus, we found
that the active concentration of mannan-rich fractions
could successfully adsorb Salmonella, a pathogen that
can cause foodborne diseases.
The following experiment examined the effects
of fractions rich in mannanooligosaccharides on the
activity, colonization, and species composition of
microflora in ducks’ intestines.
At the second (in vivo) stage, we determined the
effect of mannan-rich fractions on the number of
bacteria in the gut of experimentally infected ducks aged
30 days by type I fimbriae bacterial strains (C. jejuni
and S. enteritidis strains). In experimental groups of
birds that received prebiotic MOS fractions with feed,
the level of bacteria with type I fimbriae decreased. The
effect of mannan oligosaccharide-rich fractions on the
concentration of intestinal microflora of ducks infected
with S. enteritidis is shown in Fig. 3.
The effect of mannan-rich fractions on the
concentration of intestinal microflora of ducks infected
with C. jejuni is shown in Fig. 4.
The results showed that mannan oligosaccharides
could regulate intestinal microflora due to their selective
ability to inhibit Salmonella spp. and Campylobacter
spp. proliferation, preventing pathogenic colonization
of the intestines and minimizing its toxic effect on
the poultry. Concentration of Salmonella spp. in the
ducklings’ gut was lower by 3.69 log CFU/g and
Campylobacter spp. by 3.27 log CFU/g compared to
the control, respectively. Metabolites of functional
oligosaccharides did not affect the levels of intestinal
colonization by pathogenic bacteria (coliforms and
(a) (b) (c) (d)
Figure 2 Absorption of Salmonella enteritidis with 0.4%
concentrated pure fraction of mannan oligosaccharides
in vitro: a – in 2 min; b – in 4 min; c – in 6 min; d – in 10 min
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anoerobeds). They did not prevent Lactobacillus spp.
and Bifidobacterium spp. proliferation either, which
contributed to the colonization of beneficial bacteria in
the birds’ intestines.
Regulation of intestinal microbiocenosis can
potentially have a positive effect on immune response
mechanisms, i.e. to strengthen immunity and enhance
the poultry population.
The effect of mannan-rich fractions on the ducklings’
gut microflora infected with S. enteritidis is shown
in Fig. 4a. The reisolation rate of S. enteritidis and
C. jejuni in the test group, which received prebiotic
mannan-rich fractions with feed, decreased by 53.6 and
66.2%, respectively, compared to the control group
(Figs. 5a, 5b).
Bifido- and lactobacteria also displayed antagonistic
activity against Campylobacter jujuni and Salmonella
enteritidis isolates. It makes them possible to be used for
the prevention of infectious diseases caused by sensitive
strains of pathogens to prebiotic drugs. Bifidobacterium
spp. a nd Lactobacillus spp. suppressed the growth of
microorganisms to different extents (Table 2).
Twelve isolates (92.6%) of Campylobacter spp. were
susceptible to bifidobacteria. The inhibition zone of
campylobacter was 5.1 ± 0.3 mm. Ten Campylobacter
jujuni isolates showed a moderate level of antagonistic
activity ‒ 76.9%, with the inhibition zone of
5.1 ± 1.0 mm.
Twenty four isolates (88.9%) of S. enteritidis were
susceptible to bifidobacteria; the inhibition zone of
S. enteritidis was 5.5 ± 0.4 mm. The antagonistic
activity of lactobacilli against S. enteritidis showed a
moderate level: 22 isolates (81.5%) had inhibition zone
of 4.9 ± 0.5 mm. Bifidobacteria were more active against
Campylobacter spp. and Salmonella spp. It makes it
possible to use probiotics to prevent and treat infectious
diseases caused by susceptible strains of pathogenic
microorganisms to the drug. To improve the ducks’
productivity, we studied the effect of mannan-rich
fractions. The experiment plan is given in Table 3.
To solve the problem of bacteriosis prevention and
increase of birds’ productivity, we also studied the effect
of a combined use of mannan oligosaccharides and
probiotic bifidobacteria and lactobacilli.
Figure 3 Effects of mannan oligosaccharide-rich fractions on the concentration of intestinal microflora of ducks infected with
S. enteritidis log CFU/g
(а) (б)
5.64
6.99 6.56
9.34 9.14 9.72
7.34 7.28
8.6
9.69
0
2
4
6
8
10
12
Salmonella spp. Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
log CFU/g
Intestinal microflora
Experiment Control
5.76
6.99 6.56
9.03 9.14 9.72
7.34 7.28
8.6
9.69
0
2
4
6
8
10
12
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
log CFU/g
Intestinal microflora
Experiment Control
72.5
18.9
0
20
40
60
80
Ducks infected with S.enteritidis, %
Control Experimental
89.8
23.6
0
20
40
60
80
100
Ducks infected with C. jejuni, %
Control Experimental
(а) (б)
5.64
6.99 6.56
9.34 9.14 9.72
7.34 7.28
8.6
9.69
0
2
4
6
8
10
12
Salmonella spp. Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
log CFU/g
Intestinal microflora
Experiment Control
5.76
6.99 6.56
9.03 9.14 9.72
7.34 7.28
8.6
9.69
0
2
4
6
8
10
12
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
log CFU/g
Intestinal microflora
Experiment Control
72.5
18.9
0
20
40
60
80
Ducks infected with S.enteritidis, %
Control Experimental
89.8
23.6
0
20
40
60
80
100
Ducks infected with C. jejuni, %
Control Experimental
Figure 4 Effects of mannan oligosaccharide-rich fractions on the concentration of intestinal microflora of ducks infected with
C. jejuni log CFU/g
Lactobacillus
spp.
Bifidobacterium
spp.
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
spp.
Campylobacter
spp.
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The first stage of the experiment included 20320
ducks (Star 53 Y.Y.) divided into four groups: one control
and three experimental ones. The experiment was
carried out three times (81280 ducks in total). Probiotics
were added to the diet of the ducks with water (10 cm3
per 4 kg duck weight) once a day from the first day until
the end of the fattening period (49 days).
We added mannan-rich fractions to the base diet –
400 g/t of feed. We added bifidobacteria and mannanrich
fractions to the duck diet once a day until the end of
fattening period (49 days). The analysis showed higher
results during all periods of the birds’ life compared to
the control groups (Table 4).
At the age of 21 days, the average growth rate of
ducks receiving probiotics with mannan-rich fractions
was 87.3 g vs. to 83.6 g in the control group. We
noticed a similar trend at the age of 21 days with an
average daily growth of ducks from 101.4 g to 107.6 g.
The experimental group III after 21 days exceeded the
control group by 7.6%.
On day 21, the body weight of ducks receiving
probiotics, mannan-rich fractions, and their mix
exceeded that in the control group by 1.1, 1.9 and
3.6%, respectively. The body weight was 1273 ± 67 g,
1283 ± 42 g, and 1305 ± 34 g, respectively.
In 49 days, the body weight of the ducks receiving
mannan-rich fractions, as well as their mix was
3415 ± 95.5, 3459 ± 87.4, and 3547 ± 24.3 g, respectively,
which exceeded the weight of the ducks of the control
group by 3.1, 4.4 and 7.1 % (Table 4). In addition, a
similar trend was detected with average daily gain in
duck weight. In 49 days, it was 59.2, 59.7, and 61.3 g for
experimental groups, exceeding that in the control group
by 1.2, 2.1, and 4.7 %, respectively. The ducks receiving
the mix of probiotics and mannan-rich fractions gained
weight more intensively compared to the birds having the
other diets (Table 5).
CONCLUSION
In vitro studies showed the ability of prebiotic
mannan-rich fractions isolated from the cell walls
of Saccharomyces cerevisiae yeast to adsorb type I
fimbriae bacterial pathogens (S. enteritidis and C. jejuni)
and prevent colonization and proliferation of pathogenic
microorganisms on the surface of ducks’ intestinal
epithelial cells.
We studied the influence of fractions rich in
mannan oligosaccharides on activity, colonization,
and species composition of duck gut microflora.
(а) (б)
Figure 5 Effects of mannan-rich oligosaccharides on the reisolation rate of salmonellas from the intestines of poultry infected with
S. Enteritidis (a) and campylobacteria from the intestines of poultry infected with C. Jejuni (b)
(а) (б)
0
2
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
Intestinal microflora
Experiment Control
72.5
18.9
0
20
40
60
80
Ducks infected with enteritidis, %
Control Experimental
89.8
23.6
0
20
40
60
80
100
Ducks infected with C. jejuni, %
Control Experimental
(а) (б)
0
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
spp.
Coliforms Anaerobes
Intestinal microflora
Experiment Control
72.5
18.9
0
20
40
60
80
Ducks infected with S.enteritidis, %
Control Experimental
89.8
23.6
0
20
40
60
80
100
Ducks infected with jejuni, %
Control Experimental
(а) (б)
0
Campylobacter
spp.
Lactobacillus
spp.
Bifidobacterium
Coliforms Anaerobes
Intestinal microflora
Experiment Control
72.5
18.9
0
20
40
60
80
Ducks infected with S.enteritidis, %
Control Experimental
89.8
23.6
0
20
40
60
80
100
Ducks infected with C. jejuni, %
Control Experimental
Table 2 Susceptibility of Bifidobacterium spp. and Lactobacillus spp. (M ± m), %
Microorganism Inhibition zone, mm Control of growth
on Preston agar
Control of growth on SalmoBifidobacterium
spp. Lactobacillus spp. nella agar M1078, HiMedia
C. jujuni 5.3 ± 0.2 5.1 ± 0.3 +
S. enteritidis 5.5 ± 0.4 4.9 ± 0.5 +
(+) – signs of growth, P < 0.05
Table 3 Bifidobacteria and mannan-rich fractions in the duck’s
diet (n = 20320)
Groups Diet
Control Base diet “Starter” from day 1 to day 20 of life
Base diet “Grower” from day 21 to day 49
Experimental
group I
Base diet + probiotics from day 1 to day 49
Experimental
group II
Base diet + mannan-rich fractions from
day 1 to day 49
Experimental
group III
Base diet + probiotics + mannan-rich fractions
from day 1 to day 49
S. Enteritidis, % C. Jejuni, %
344
Kasjanenko S.M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 337–347
S. enteritidis reisolation rate decreased by 53.6% and
C. jejuni ‒ by 66.2% in ducks receiving fractions rich
in mannanooligosaccharides, compared to the control
group. Experiments showed that the addition of prebiotic
fractions to the diet did not affect the concentration of
lactobacilli, bifidobacteria, enterococci, and anaerobic
bacteria.
Bifido- and lactobacteria have antagonistic
activity against circulating strains of S. enteritidis and
C. jejuni. 88.9% of S. enteritidis isolates were
susceptible to bifidobacteria and 81.5% of the studied
strains were susceptible to lactobacilli. 92.6% of the
isolated Campylobacter jejuni were susceptible to
bifidobacteria, 76.9% of Campylbacter strains were
susceptible to lactobacteria.
We developed a method of preventing bacterial
infections and increasing ducks’ productivity based
on the combined use of bifido- and lactobacteria
(1.5×109 CFU/mL) in a ratio of 1:1 with water and
fractions enriched with mannan oligosaccharides
(0.4 kg/t) together with feed. We recommend the
preparation from the first day of birds’ life till the end of
growing period.
Preventive measures improved the preservation
of the duck population by 8.76%, ensuring the average
daily increase by 6.9% and the reduction of feed costs by
Table 4 Effect of experimental diets on duck growth (M ± m)
Indexes Groups
Control
(base diet)
Experimental
group I
(diet with
probiotics)
Experimental
group II (diet
with mannanrich
fractions)
Experimental
group III (diet with
probiotics and mannan-
rich fractions)
Days 0–21
number of birds on day 1 20320 20320 20320 20320
average body weight of ducks, g
average body weight of ducks,%
1259 ± 45*
100
1273 ± 67*
101.1
1283 ± 42*
101.9
1305 ± 34*
103.6
average daily gain of ducks, g
average daily gain of ducks,%
83.6 ± 8.4
100
84.8 ± 8.1
101.4
85.7 ± 9.5
102.5
87.3 ± 8.2
107.6
safety of poultry,% 91.3 92.4 93.46 104.4
Days 22–49
number of birds on day 22 18552 18703 18991 19537
average body weight of ducks, g
average body weight of ducks,%
3312 ± 35.3*
100
3415 ± 95.5*
103.1
3459 ± 87.4*
104.4
3547 ± 24.3*
107.1
average daily gain of ducks, g
average daily gain of ducks,%
58.5
100
59.2
101.2
59.7
102.1
61.3
104.7
number of birds on day 49, heads
number of birds on day 49, %
16537
89.14
17353
92.78
18060
95.10
19127
97.9
cost of feed 97644.3 103216.7 104128.4 105331.7
feed consumption per 1 kg of growth for 49 days, kg
feed consumption per 1 kg of growth for 49 days, %
2.01
100.0
1.91
91.52
1.88
93.53
1.86
95.02
*The values in the column for each treatment stage that does not share the overall upper index vary significantly (P < 0.05). Each value is an
average of n = 3 flocks per diet with 36, 30 and 30 birds in the flock for each growing period, respectively. Comparisons between the groups were
made using the Tukeys HSD test, P < 0.05 we considered statistically significant
Table 5 Average body weight of ducks receiving probiotics and mannan-rich fractions during different periods of growth
and development, g/head (n = 50)
Age,
weeks
Groups
Control (base diet) Experimental group I
(diet with probiotics)
Experimental group II
(diet with mannan-rich
fractions)
Experimental group III
(diet with probiotics and
mannan-rich fractions)
Standard
values
0 52.35 ± 0.57* 52.64 ± 0.37* 53.22 ± 0.67* 52.66 ± 0.81* 52
1 205.52 ± 1.43* 206.53 ± 0.58* 208.42 ± 0.37* 215.53 ± 0.48* 206
2 640.37 ± 2.93* 642.34 ± 3.25* 645.38 ± 5.34* 678.59 ± 13.73* 645
3 1239.17 ± 5.52* 1247.72 ± 5.51* 1258.42 ± 14.53* 1305.48±34.27* 1257
4 1814.58 ± 7.74* 1874.25 ± 22.47* 1883.58 ± 11.43* 1933.53 ± 31.45* 1876
5 2351.34 ± 33.34* 2404.43 ± 27.48* 2486.35 ± 42.28* 2592.63 ± 47.81* 2503
6 2918.42 ± 27.56* 2948.27 ± 25.58* 2915.37 ± 33.59* 3197.37 ± 49.56* 3100
7 3319.68 ± 26.85* 3419.62 ± 24.37* 3528.63 ± 25.57* 3683.87±25.79* 3500
345
Kasjanenko S.M. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 337–347
4.98% for 1 kg of growth throughout the growing period.
During the experiment, we recorded a significant
decrease in Salmonella and Campylobacter colonization
in the poultry intestines and improved average daily
growth. The biologically active supplements provided a
significant advantage in industrial duck farming.
We demonstrated the effectiveness of natural and
environmentally safe methods: yeast fractions rich in
mannan oligosaccharides, probiotics, and their combined
use. The method was effectively implemented in
Ukrainian poultry farms.
CONTRIBUTION
Concept – O.I. Kasjanenko, L.V. Nagornaya,
S.M. Kasjanenko; Design – V.V. Melnychuk,
S.M. Kasjanenko; Observation – O.I. Kasjanenko,
V.A. Yevstafieva; Resources – S.M. Kasjanenko;
Materials – V.A. Yevstafieva, L.V. Nagornaya,
S.M. Kasjanenko; Data collection and/or processing –
S.M. Kasjanenko, O.I. Kasjanenko, L.V. Nagornaya,
V.A. Yevstafieva; Analysis and/or interpretation –
O.I. Kasjanenko, L.V. Nagornaya, V.V. Melnychuk,
V.A. Yevstafieva; Search for literature –
O.I. Kasjanenko, S.M. Kasjanenko, V.V. Melnychuk,
G.A. Lukyanova, I.A. Gurenko; Writing the manuscript
– O.I. Kasjanenko, L.V. Nagornaya, S.M. Kasjanenko,
G.A. Lukyanova, I.A. Gurenko; Critical review –
O.I. Kasjanenko, G.A. Lukyanova, I.A. Gurenko.
CONFLICT OF INTEREST
The authors have no conflict of interest.
ACKNOWLEDGEMENTS
The authors thank the managers of the poultry farms
for their assistance in conducting experiments.

1. Kittler S, Fischer S, Abdulmawjood A, Glunder G, Klein G. Colonisation of a phage susceptible Campylobacter jejuni population in two phage positive broiler flocks. PLoS One. 2014;9(4). DOI: https://doi.org/10.1371/journal.pone.0094782.

2. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks breaks in 2015. EFSA Journal. 2016;14(12). DOI: https://doi.org/10.2903/j.efsa.2016.4634.

3. Kasyanenko OI, Fotina TI, Fotina АА, Gladchenko SM, Gnidenko TY, Bezruk RV. Properties of Сampylobacter jejunі that were isolated from poultry products. Microbiological Journal. 2017;79(4):66-74. (In Russ.).

4. Umaraw P, Prajapati A, Verma AK, Pathak V, Singh VP. Control of campylobacter in poultry industry from farm to poultry processing unit: A review. Critical Reviews in Food Science and Nutrition. 2017;57(4):659-665.

5. Collard JM, Bertrand S, Dierick K, Godard C, Wildemauwe C, Vermeersch K, et al. Drastic decrease of Salmonella Enteritidis isolated from humans in Belgium in 2005, shift in phage types and influence on foodborne outbreaks. Epidemiology and Infection. 2008;136(6):771-781. DOI: https://doi.org/10.1017/s095026880700920x.

6. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA Journal. 2015;13(12). DOI: https://doi.org/10.2903/j.efsa.2015.4329.

7. Meunier M, Guyard-Nicodeme M, Dory D, Chemaly M. Control strategies against Campylobacter at the poultry production level: biosecurity measures, feed additives and vaccination. Journal of Applied Microbiology. 2016;120(5):1139-1173. DOI: https://doi.org/10.1111/jam.12986.

8. Skarp CPA, Hanninen ML, Rautelini HIK. Campylobacteriosis: the role of poultry meat. Clinical Microbiology and Infection. 2016;22(2):103-109. DOI: https://doi.org/10.1016/j.cmi.2015.11.019.

9. Dogan ANC, Celik E, Kilicle PA, Atalay E, Saglam AG, Dogan A, et al. Antibacterial effect of hot peppers (Capsicum annuum, Capsicum annuum var globriusculum, Capsicum frutescens) on some Arcobacter, Campylobacter and Helicobacter species. Pakistan Veterinary Journal. 2018;38(3):266-270. DOI: https://doi.org/10.29261/pakvetj/2018.057.

10. Spring P, Wenk C, Connolly A, Kiers A. A review of 733 published trials on Bio-Mos®, a mannan oligosaccharide, and Actigen®, a second generation mannose rich fraction, on farm and companion animals. Journal of Applied Animal Nutrition. 2015;3. DOI: https://doi.org/10.1017/jan.2015.6.

11. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2016. EFSA Journal. 2017;15(12). DOI: https://doi.org/10.2903/j.efsa.2017.5077.

12. Ansari F, Pourjafar H, Bokaie S, Peighambari SM, Mahmoudi M, Fallah MH, et al. Association between poultry density and Salmonella infection in commercial laying flocks in Iran using a Kernel density. Pakistan Veterinary Journal. 2017;37(3):299-304.

13. Gormley FJ, Bailey RA, Watson KA, McAdam J, Avendaño S, Stanley WA, et al. Campylobacter colonisation and proliferation in the broiler chicken under natural field challenge is not affected by bird growth rate or breed. Applied and Environmental Microbiology. 2014;80(21):6733-6738. DOI: https://doi.org/10.1128/AEM.02162-14.

14. Ao Z, Choct M. Oligosaccharides affect performance and gut development of broiler chickens. Asian-Australasian Journal of Animal Sciences. 2013;26(1):116-121. DOI: https://doi.org/10.5713/ajas.2012.12414.

15. Ramirez-Hernandez A, Rupnow J, Hutkins RW. Adherence reduction of Campylobacter jejuni and Campylobacter coli strains to HEp-2 Cells by mannan oligosaccharides and a high-molecular-weight component of cranberry extract. Journal of Food Protection. 2015;78(8):1496-1505. DOI: https://doi.org/10.4315/0362-028X.JFP-15-087.

16. Muller KH, Collinson SK, Trust TJ, Kay WW. Type-1 fimbriae of Salmonella-Enteritidis. Journal of Bacteriology. 1991;173(15):4765-4772.

17. Lea H, Spring P, Taylor-Pickard J, Burton E. Natural carbohydrate fraction Actigen™ from Saccharomyces cerevisiae cell wall: effects on goblet cells, gut morphology and performance of broiler chickens. Journal of Applied Animal Nutrition. 2012;1. DOI: https://doi.org/10.1017/jan.2013.6.

18. Rous P, Turner JR. The preservation of living red blood cells in vitro: I. Methods of preservation. Journal of Experimental Medicine. 2016;23(2):219-237. DOI: https://doi.org/10.1084/jem.23.2.219.

19. Corrigan A, de Leeuw M, Penaud-Frezet S, Dimova D, Murphy RA. Phylogenetic and functional alterations in bacterial community compositions in broiler ceca as a result of mannan oligosaccharide supplementation. Applied and Environmental Microbiology. 2015;81(10):3460-3470. DOI: https://doi.org/10.1128/aem.04194-14.

20. Arsi K, Donoghue AM, Woo-Ming A, Blore PJ, Donoghue DJ. The efficacy of selected probiotic and prebiotic combinations in reducing Campylobacter colonization in broiler chickens. Journal of Applied Poultry Research. 2015;24(3):327-334. DOI: https://doi.org/10.3382/japr/pfv032.

21. Hadadji M, Benama R, Saidi N, Henni DE, Kihal M. Identification of cultivable Bifidobacterium species isolated from breast-fed infants feces in West-Algeria. African Journal of Biotechnology. 2005;4(5):422-430.

22. Spring P, Wenk C, Dawson KA, Newman KE. The effects of dietary mannaoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of salmonella-challenged broiler chicks. Poultry Science. 2000;79(2):205-211. DOI: https://doi.org/10.1093/ps/79.2.205.

23. Pitkanen T. Review of Campylobacter spp. in drinking and environmental waters. Journal of Microbiological Methods. 2013;95(1):39-47. DOI: https://doi.org/10.1016/j.mimet.2013.06.008.

24. Ugarte-Ruiz M, Florez-Cuadrado D, Wassenaar TM, Porrero MC, Dominguez L. Method comparison for enhanced recovery, isolation and qualitative detection of C. jejuni and C. coli from wastewater effluent samples. International Journal of Environmental Research and Public Health. 2015;12(3):2749-2764. DOI: https://doi.org/10.3390/ijerph120302749.

25. Sujatha K, Dhanalakshmi K, Rao AS. Antigenic characterisation and antibiotic sensitivity of field isolates of Salmonella gallinarum. Indian Veterinary Journal. 2003;80(10):965-968.

26. Mannan SJ, Rezwan R, Rahman MS, Begum K. Isolation and biochemical characterization of Lactobacillus species from yogurt and cheese samples in Dhaka metropolitan area. Bangladesh Pharmaceutical Journal. 2017;20(1):27-33.

27. Zinedine A, Faid M. Isolation and characterization of strains of bifidobacteria with probiotic proprieties in vitro. World Journal of Dairy and Food Sciences. 2007;(1):28-34.

28. Achtman M, Wain J, Weill FX, Nair S, Zhou ZM, Sangal V, et al. Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica. PLoS Pathogens. 2012;8(6). DOI: https://doi.org/10.1371/journal.ppat.1002776.

29. Baurhoo B, Ferket PR, Zhao X. Effects of diets containing different concentrations of mannanoligosaccharide or antibiotics on growth performance, intestinal development, cecal and litter microbial populations, and carcass parameters of broilers. Poultry Science. 2009;88(11):2262-2272. DOI: https://doi.org/10.3382/ps.2008-00562.

30. Rakibul Hasan AKM, Ali MH, Siddique MP, Rahman MM, Islam MA. Сlinical and laboratory diagnoses of common bacterial diseases of broiler and layer chickens. Bangladesh Journal of Veterinary Medicine. 2010;8(2):107-115. DOI: https://doi.org/10.3329/bjvm.v8i2.11188.

31. Srinivasan P, Balasubramaniam GA, Murthy T, Saravanan S, Balachandran P. Prevalence and pathology of Salmonellosis in commercial layer chicken from Namakkal, India. Pakistan Veterinary Journal. 2014;34(3):324-328.

32. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA Journal. 2018;16(12). DOI: https://doi.org/10.2903/j.efsa.2018.5500.

33. Safaei HG, Jalali M, Hosseini A, Narimani T, Sharifzadeh A, Raheimi E. The prevalence of bacterial contamination of table eggs from retails markets by Salmonella spp., Listeria monocytogenes, Campylobacter jejuni and Escherichia coli in Shahrekord, Iran. Jundishapur Journal of Microbiology. 2011;4(4):249-253.

34. Jamshidi A, Kalidari GA, Hedayati M. Isolation and identification of Salmonella Enteritidis and Salmonella Typhimurium from the eggs of retail stores in Mashhad, Iran using conventional culture method and multiplex PCR assay. Journal of Food Safety. 2010;30(3):558-568. DOI: https://doi.org/10.1111/j.1745-4565.2010.00225.x.

35. Khoult D, Krig N, Snit P. Opredelitelʹ bakteriy Berdzhi. Tom 1 [Bergey`s Manual of Determinative Bacteriology. Vol. 1]. Moscow: Mir; 1997. 432 p. (In Russ.).