Effects of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid and

their combination on growth performance and intestinal health in young

broiler chickens challenged with Salmonella Typhimurium

V. Jazi,∗ A. D. Foroozandeh,† M. Toghyani,† B. Dastar,∗ R. Rezaie Koochaksaraie,∗ and M. Toghyani‡,1

∗
Department of Animal and Poultry Nutrition, Faculty of Animal Science, Gorgan University of Agricultural
Sciences and Natural Resources, Gorgan, Iran; † Department of Animal Science, Isfahan (Khorasgan) Branch,
Islamic Azad University, Isfahan, Iran; and ‡ Department of Animal Science, School of Environmental and Rural
Science, University of New England, Armidale, NSW 2351, Australia

ABSTRACT This study compared the efficacy of Pe-
diococcus acidilactici, mannan-oligosaccharide, butyric
acid, and their combination on growth performance
and intestinal health in broiler chickens challenged
with S. Typhimurium. Ross 308 male broilers (n =
420) were randomly assigned to one of the 6 treat-
ments, resulting in 5 replicate pens of 14 chicks per
treatment. The treatments included a negative con-
trol [(NC), no additive, not challenged]; positive con-
trol [(PC), no additive, but challenged with S. Ty-
phimurium at d 3 posthatch], and 4 groups whereby
birds were challenged with S. Typhimurium at d 3
posthatch and fed diets supplemented with either pro-
biotic [0.1 g/kg Pediococcus acidilactici (PA)], prebi-
otic [2 g/kg mannan-oligosaccharides (MOS)], organic
acid [0.5 g/kg butyric acid (BA)], or a combination
of the 3 additives (MA). The S. Typhimurium chal-
lenge decreased feed intake, body weight gain and in-
creased feed conversion ratio and reduced jejunum vil-
lus height (VH) and VH to crypt depth (CD) ratio (P
Key words: Salmonella Typhimurium, broiler chickens, digestive enzymes, cecal microbiota, gut morphology
< 0.05). Birds on the MA treatment exhibited simi-
lar performance to birds on the NC treatment (P >
0.05) and had a lower population of Salmonella in the
ceca compared with birds on the PC treatment, at
d 14 and 21 post-challenge (P < 0.05). The lowest
heterophil to lymphocyte ratio was observed in birds
on the MA and NC treatments (P < 0.05). Birds fed
diets supplemented with MA or PA had greater VH
and VH: CD ratio than birds on the PC treatment at
d 7, 14 and 21 d post-challenge (P < 0.05). Suppressed
amylase and protease activity was observed as a result
of the S. Typhimurium challenge; the enzyme levels
were restored in birds fed the additive-supplemented
diets, when compared to the birds on the PC treat-
ment, particularly at d 21 post-challenge (P < 0.05).
These results indicate that dietary supplementation
with a combination of PA, BA, and MOS in broiler
chickens could be used as an effective tool for con-
trolling S. Typhimurium and promoting growth per-
formance.
2018 Poultry Science 97:2034–2043
http://dx.doi.org/10.3382/ps/pey035

INTRODUCTION of the animal products (Cheng et al., 2014). However,

Salmonella enterica var. Typhimurium (S. Ty-
phimurium) is one of the most prevalent serotypes
of Salmonella, a key causative agent of salmonel-
losis, which is a disease that enters the human
food chain through animal products, particularly raw
poultry products (Chalghoumi et al., 2009; Thung
et al., 2016). In-feed antibiotics have long been used
at sub-therapeutic doses in poultry feed to control gut
pathogenic bacteria load and hence to enhance feed effi-
ciency, promote animal growth, and improve the quality

C 2018 Poultry Science Association Inc.
Received August 28, 2017.
Accepted January 13, 2018.
1
Corresponding author: Mtoghya2@une.edu.au
the increased risk of bacteria acquiring resistance to an-
tibiotics and their residue in end-products such as meat
and eggs led to a ban on the use of in-feed antibiotics
as growth promotors (AGP) more than a decade ago
in many countries in the European Union (Toghyani
et al., 2010; Sugiharto, 2016). As a result of this grow-
ing pressure on livestock producers worldwide, various
alternative strategies have been proposed and investi-
gated as potential substitutes for AGPs in animal feeds.
Particular attention has been paid to the use of probi-
otics (Menconi et al., 2011; Park and Kim, 2014), pre-
biotics (Pourabedin et al., 2016; Rajani et al., 2016),
and organic acids (Fernandez-Rubio et al., 2009; Saleem
et al., 2016), as AGPs alternatives in poultry feed.
There is evidence to suggest that these aforemen-
tioned products exert beneficial effects on performance

2034
 SALMONELLA CHALLENGE AND FEED ADDITIVES 2035
parameters, microbiota composition, and intestinal in- Table 1. Compositions of the experimental diets.
tegrity, and potentially reduce the levels of pathogenic
% as-is
bacteria such as Salmonella in the tract. However, these
Ingredient Starter (d 1–10) Grower (d 11–24)
positive effects have not always been consistent (Van
der Aar et al., 2017). Corn 55.5 65.5
The main proposed modes of action of probiotics in- Soybean meal 37.7 28.1
Sunflower oil 2.37 2.24
clude 1) antagonistic action towards pathogenic bac- Limestone 1.09 0.99
teria, by secreting products that inhibit their develop- Dicalcium phosphate1 1.74 1.54
ment, such as bacteriocins, organic acids, and hydrogen Sodium chloride 0.21 0.18
Sodium bicarbonate 0.16 0.19
peroxide, and 2) competitive exclusion, by competing Vitamin premix2 0.25 0.25
with bacteria for locations in the intestinal mucous Mineral premix3 0.25 0.25
membrane to adhere to and nutrients (Patterson and Choline chloride 60% 0.122 0.116
DL-methionine 0.296 0.254
Burkholder, 2003). Prebiotics serve as a substrate for L-lysine 0.188 0.269
endogenous beneficial bacteria, thus promoting com- L-threonine 0.091 0.079
petitive exclusion of pathogenic microbes and selective Nutrient composition
colonization by beneficial microbes (Biggs et al., 2007). Metabolizable energy (Kcal/kg) 3000 3100
Crude protein (%) 22.94 19.41
Oligosaccharides display prebiotics properties, but they Dig4 lysine (%) 1.28 1.11
have also been reported to present immunomodulatory Dig methionine (%) 0.63 0.55
beneficial effects in the gut such as modifying clear- Dig methionine + cysteine (%) 0.95 0.83
Dig threonine (%) 0.86 0.72
ance efficiency of pathogenic bacteria, activating T cell- Calcium (%) 0.90 0.80
dependent immune responses and repression of pro- Available phosphorus (%) 0.45 0.40
inflammatory cytokines (Troy and Kasper, 2010; Bonos Sodium (%) 0.16 0.17
Chloride (%) 0.21 0.21
et al., 2011). Organic acids, such as lactic, acetic, bu- Choline (mg/kg) 1700 1500
tyric, tannic, fumaric and propionic acids, display an-
1
timicrobial properties and play a crucial role in con- Dicalcium phosphate contained: phosphorus, 18%; calcium, 21%.
2
Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate, 0.02 mg
trolling the population of pathogenic bacteria (Menconi cholecalciferol, 8.3 mg alphatocopheryl acetate, 2.2 mg menadione, 2 mg
et al., 2014), likely by lowering gastrointestinal tract pyridoxine HCl, 8 mg cyanocobalamin,10 mg nicotine amid, 0.3 mg folic
pH (Partanen and Mroz, 1999) and stimulating protein acid, 20 mg D-biotin and 160 mg choline chloride.
3
Supplied per kg of diet: 32 mg Mn (MnSO4 ·H2 O), 16 mg Fe
digestion by converting pepsinogen to pepsin (Suiryan- (FeSO4 ·7H2 O), 24 mg Zn (ZnO), 2 mg Cu (CuSO4 ·5H2 O), 800 μ g I
rayna and Ramana, 2015). (KI), 200 μ g Co (CoSO4 ) and 60 μ g Se.
4
The differing mechanisms and modes of action ex- Dig, Digestible.
hibited by probiotics, prebiotics, and organic acids sug-
gest that there could be a complementary synergistic ef- were fed a common starter diet for the first 3 d. At
fect resulting from supplementing diets with a mixture d 3 posthatch, birds were weighed and randomly as-
of these additives, especially under the stressful con- signed to one of the 6 experimental treatments with 5
ditions imposed by a S. Typhimurium challenge (Ra- replicate pens of 14 birds each to have a starting body
jani et al., 2016). In view of an evident dearth of data weight of 72 ± 1.5 g/bird. The treatments consisted
in this field, the current study was designed to deter- of negative control [(NC), no additive and no chal-
mine the efficacy of Pediococcus acidilactici (a species lenge with S. Typhimurium]; positive control [(PC),
of gram-positive cocci that produces lactic acid and se- no additive, but challenged with S. Typhimurium], and
crets a bacteriocins known as pediocins) as a probi- 4 groups whereby birds were challenged with S. Ty-
otic supplement, mannan oligosaccharides as a prebi- phimurium and fed diets supplemented with either pro-
otic supplement, and micro-encapsulated butyric acid, biotic (Pediococcus acidilactici at 0.01% [Pedi Guard,
singularly or in combination, on growth performance Iran, concentration 1 × 1010 CFU/g]; PA), prebiotic
and gut health of young broiler chicks challenged with (0.2% mannan-oligosaccharides, [ActiveMOS R
, Biori-
S. Typhimurium. gin, Brazil]; MOS), butyric acid (0.05% butyric acid
provided in an encapsulated form consisting of 50% bu-
tyrate salt [ButiPEARL; Kemin Industries, Herentals,
MATRIALS AND METHODS Belgium]; BA), or a combination of the 3 additives
Experimental Design, Dietary Treatments, (MA). All the challenged birds were orally adminis-
tered 105 CFU of S. Typhimurium at d 3 posthatch.
and Bird Husbandry Birds were allowed ad libitum access to the treatment
The experimental procedures in this study were re- diets and water for the duration of the trial (d 0 to 24).
viewed and approved by the Animal Ethics committee A basal corn-SBM diet was formulated to meet the nu-
of Islamic Azad University, Isfahan Branch, based on trient requirements according to the Ross 308 (Aviagen,
institutional and national guidelines for the care and 2014) for starter (0- to 10-d) and grower (10- to 24-d)
use of animals. A total of 420 day-old Ross 308 male periods (Table 1). The temperature and lighting pro-
broiler chicks were obtained from a commercial hatch- grams used followed the Ross 308 breed management
ery (Navid Morgh Guilan Company., Guilan, Iran), recommendations (Aviagen, 2014).
2036 JAZI ET AL.
Bacterial Inoculum Preparation
The S. Typhimurium strain (PTCC 1709) was ob-
tained as a freeze-dried powder from the Iranian
Research Organization for Science and Technology,
Tehran. The frozen bacterial culture was thawed and
cultured in a trypticase soy broth for 24 at 37◦ C. The
number of CFU was determined by diluting the inocu-
lum with sterile phosphate-buffered saline (PBS) and
plating it on culture medium XLT4 agar for 24 h at
37◦ C. At d 3 posthatch, all birds (except those in the
NC treatment group) were orally administered a one-
off dose of 0.5 mL of 1 × 105 CFU S. Typhimurium
suspension. The same dose per os of sterile PBS was
orally administered to the birds in unchallenged group.
Performance Measurements
Growth performance parameters including feed in-
take (FI) and body weight gain (BWG) were measured
during the starter (d 0 to 7 post challenge), grower
(d 7 to 21 post challenge), and overall experimental
period (d 0 to 21 post challenge), and used to calculate
the feed conversion ratio (FCR), adjusted for mortal-
ity. Ten birds per treatment (2 birds from each replicate
pen) were randomly selected for blood, digesta and tis-
sue sample collection at d 7, 14, and 21 post-challenge.
Hematological Parameters
At d 7, 14, and 21 post-challenge, 3 mL of blood was
obtained from the 2 birds per pen by puncturing the
brachial vein and individually collecting the blood into
non-heparinized tubes. Blood smears were prepared by
the May-Grunwald-Giemsa coloring technique and het-
erophil to lymphocyte (H/L) ratio was calculated by
counting 100 leukocytes per slide.
Enumeration of Cecal Bacteria
Following blood sample collection, the 2 birds were
weighed and euthanized by CO2 asphyxiation. The con-
tents of the ceca were collected by gently squeezing the
digesta into plastic containers, and pooled per replicate.
To determine S. Typhimurium counts, 1 g of the ceca
digesta was diluted with 0.1% peptone water, homoge-
nized for 1 min and serial dilutions were subsequently
prepared in the same diluent. Samples at the optimum
dilution level were then selected and 0.1 mL was plated
in triplicate on XLT4 Agar (Difco) containing 100 mL
of Nalidixic acid. The plates were then incubated for
24 h at 37◦ C under anaerobic conditions.
To determine Lactobacillus, Bifidobacterium, and co-
liform counts in the ceca samples, again 10-fold serial
dilutions were conducted with peptone water and then
0.1 mL of sample from the optimum dilution level was
cultured on plates containing modified Rogosa SL agar,
TOS Propionate agar, and MacConkey agar, respec-
tively. Plates were incubated in anaerobic conditions for
24 to 48 h at 37◦ C. Following incubation, the number
of colonies on each plate was counted and the obtained
numbers were multiplied by reversed dilution, and re-
ported as the number of CFU per 1 g sample.
Intestinal Morphology
The digesta content of the jejunum from the 2 birds
was gently flushed out with ultra-pure water into a con-
tainer, and then approximately 3 cm of the jejunum
tissue (middle section) was transected and immersed in
10% buffered formalin. Tissues were serial dehydrated
by passing the tissue through increasing concentrations
of ethyl alcohol, before being embedded in paraffin wax.
Histological examinations were carried out according to
the method described by Xu et al. (2003). A total of 10
well-oriented, intact villi and crypts were randomly se-
lected in duplicate from each tissue sample and the av-
erage of 20 values were obtained for each bird. All mor-
phological parameters were measured using the ImageJ
software package (http://rsb.info.nih.gov/ij/).
Enzyme Activity
The collected jejunum digesta samples were diluted
10 times with phosphate- saline buffer, based on weight,
and were then centrifuged at 18,000 × g for 20 min
at 4◦ C. The supernatant was stored at −80◦ C prior to
enzyme analysis. Samples were processed to determine
amylase (EC 3.2.1.1), lipase (EC 3.1.1.3), and neutral
protease activities following the methods described by
Xu et al. (2003). One unit of amylase activity was de-
fined as the amount of amylase required to release 1 mg
of glucose after 30 min incubation at 40◦ C, per mg of in-
testinal digesta protein. Lipase activity was equal to the
volume (in mL) of 0.05 M NaOH required to neutralize
the fatty acids liberated during a 6-h incubation with
3 mL of lipase substrate at 38◦ C, per mg of intestinal
digesta protein. The protease activity unit was defined
as milligrams of azocasein degraded after 2 h incubation
at 37◦ C, per mg of intestinal digesta protein.
Statistical Analysis
Firstly, Kolmogorov-Smirnov testing was conducted
to confirm normality of the data. Data were then sub-
jected to one-way analysis of variance (ANOVA) us-
ing General Linear Model procedure of SAS 9.3 package
(SAS Institute Inc., 2010) to determine the equality of
the means. Each pen was considered as an experimen-
tal unit. The values presented in the tables represent
the mean for each treatment, with a pooled standard
error of mean (SEM). Tukey’s HSD test was used to
make pairwise comparisons between means, where ap-
propriate. Statistical significance was declared at P <
0.05.
 SALMONELLA CHALLENGE AND FEED ADDITIVES 2037
Table 2. Effects of dietary treatments on growth performance of broilers.

Treatments1
2
Item NC PC PA MOS BA MA SEM P-value

Day 3
BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88
0 to 7 d P-Ch
BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 < .001
FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03
FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001
7 to 21 d P-Ch
BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 < .001
FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03
FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001
0 to 21 d P-Ch
BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 < .001
FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02
FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 < .001
a-c
Means with different superscripts in each row are significantly different (P < 0.05).
1
NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P.
acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA,
butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.
2
P-Hatch, Posthatch; P-Ch, Post-Challenge; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio.

RESULTS Bifidobacterium count compared to both the PC and

Bird’s Performance and Mortality Rate
The effects of the treatments on bird performance
are reported in Table 2. The S. Typhimurium challenge
decreased FI, BWG, and increased FCR in birds in the
PC treatment group compared to the NC treatment
group, at all time periods tested (P < 0.05). Dietary
supplementation of PA, MOS, BA, and in particular
their combination, improved FI, BWG, and FCR in the
challenged birds, compared to birds in the PC treat-
ment group (P < 0.05), at all measured time periods.
However, only birds in the MA treatment group dis-
played a body weight gain (0 to 21 d post challenge)
that was statistically similar to birds in the NC treat-
ment group (P > 0.05); birds fed the diets containing
just one of the dietary additives had a lower BWG and
higher FCR compared to birds on the NC treatment
(P < 0.05). No significant effect of challenge or dietary
treatment was observed on bird mortality during the
experimental period (data not shown).
Characterization of Cecal Microbiota
As illustrated in Figure 1, birds in the PC treat-
ment group had the lowest ceca Lactobacillus popula-
tion compared to all other treatments, at d 14 post-
challenge (P < 0.05). Birds in the treatment groups
with dietary supplementation of PA, MOS, BA and MA
had a greater population of cecal lactobacilli compared
to birds in the PC or NC treatment groups at d 14 and
21 post-challenge (P < 0.05), with the highest pop-
ulation observed in birds on the MA treatment. The
cecal population of Bifidobacterium was not affected
by the challenge (P > 0.05); however, dietary supple-
mentation with MOS and MA significantly increased
NC treatment groups at d 21 post-challenge (P < 0.05;
Figure 2). The S. Typhimurium challenge had no im-
pact on the population of coliforms in the ceca (P >
0.05, Figure 3). Birds on the NC and PC treatments
had the highest population of coliforms at d 7 and 21
post-challenge, whilst birds in the MA treatment group
had the lowest population (P < 0.05; Figure 3). No
Salmonella spp. were detected in the ceca of the un-
challenged birds. Birds fed the dietary supplements had
lower prevalence of Salmonella in the ceca compared to
birds in the PC group at d 14 and 21 post-challenge
(P < 0.05; Figure 4), and birds on the MA treatment
had lower ceca Salmonella content compared to birds
on any other treatment (P < 0.05; Figure 4).
Hematological Parameters, Intestinal
Enzyme Activities and Morphological
Analysis
Table 3 summarizes the effect of dietary treatments
on heterophil and lymphocyte counts and the H/L ra-
tio, measured at d 7, 14, and 21 post-challenge. An
increased heterophil count at d 7 and 21 post-challenge
and greater H/L ratios at all the periods tested were ob-
served with the presence of the S. Typhimurium chal-
lenge (P < 0.05). The challenged birds fed the diets
supplemented with PA, MOS, BA, and MA had signif-
icantly reduced heterophil and increased lymphocyte
counts at d 7, 14, and 21 post-challenge compared to
birds in the PC treatment group (P < 0.05). There were
no significant differences between birds in the NC and
MA treatment groups with regards to the H/L ratio at
d 14 and 21 post-challenge. Heterophil counts at d 14
and lymphocyte counts at d 21 post-challenge were not
influenced by the treatments (P > 0.05).
2038 JAZI ET AL.

Figure 1. Effects of dietary treatments on cecal Lactobacillus population in broiler chicks at 7 to 14 and 21 d after challenge with S.
Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative
control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge
with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S.
Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.

Figure 2. Effects of dietary treatments on cecal Bifidobacterium population in broiler chicks at 7 to 14 and 21 d after challenge with S.
Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative
control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge
with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S.
Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.

As illustrated in Table 4, S. Typhimurium challenge
significantly decreased amylase and protease activity
measured in the jejunum at d 7, 14, and 21 post-
challenge, and lipase activity at d 7 post-challenge (P
< 0.05). Amylase and protease activity in birds fed the
diets supplemented with PA, MOS, BA, or MA was
either higher or equal to that measured in birds on
the PC treatment group at all ages studied, and was
consistently higher in birds fed the supplemented diets
compared to birds on the NC treatment at d 21 post-
challenge (P < 0.05). Additionally, at d 7 post-challenge
there was no significant difference in lipase activity ob-
served between birds on the NC treatment and those
fed the supplemented diets (P > 0.05).
As demonstrated in Table 5, the S. Typhimurium
challenge decreased jejunum villus height (VH) in birds
on the PC treatment at d 14 and 21 post-challenge (P
< 0.05). Birds fed the supplemented diets had signifi-
cantly greater VH compared to birds on the PC treat-
ment (P < 0.05), and birds fed the diets supplemented
with PA or MA presented VH that was similar to that
observed in birds on the NC treatment (P > 0.05).
The dietary treatments had no significant impact on
crypt depth (CD; P > 0.05), but at d 7 lower VH: CD
 SALMONELLA CHALLENGE AND FEED ADDITIVES 2039

Figure 3. Effects of dietary treatments on cecal Coliforms population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium.
Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control
(not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S.
Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium;
MA, a mixture of additives + challenge with S. Typhimurium.

Figure 4. Effects of dietary treatments on cecal Salmonella population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium.
Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control
(not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S.
Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium;
MA, a mixture of additives + challenge with S. Typhimurium.

ratios were observed in birds fed the supplemented diets
compared to those on the PC treatment. At d 14 and
21 there was no significant difference between birds on
the NC treatment and those fed the supplemented diets
(P > 0.05), except for birds fed the diets supplemented
with BA.
DISCUSSION
Enteric microbiota play an important role in dic-
tating health and consequently growth responses in
broiler chickens (Van der Aar et al., 2017), but there
is a concern that pathogenic bacteria in this micro-
biota may contaminate poultry meat during process-
ing, resulting in foodborne diseases such as salmonel-
losis. Reducing Salmonella load in live animals is con-
sidered to be the most effective strategy for reduc-
ing this contamination and thus the number of human
salmonellosis cases (EFSA, 2004). The current study
investigated the efficacy of probiotic, prebiotic, butyric
acid and their mixture on diminishing Salmonella load,
by evaluating their impact on growth performance, in-
testinal parameters, and cecal digesta microbiota com-
positions in broiler chickens orally challenged with S.
Typhimurium.
2040 JAZI ET AL.
Table 3. Effects of dietary treatments on heterophil and lymphocyte counts.

Treatments1
2
Item Day P-Ch NC PC PA MOS BA MA SEM P-value

Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 < .001
14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34
21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001
Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002
14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04
21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13
H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 < .001
14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005
21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 < .001
a-c
Means with different superscripts in each row are significantly different (P < 0.05).
Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point.
1
NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P.
acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA,
butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.
2
P-Ch, Post-Challenge.

Table 4. Effects of dietary treatments on intestinal digestive enzyme activities (U/mg of digesta protein).

Treatments1
2
Enzymes Day P-Ch NC PC PA MOS BA MA SEM P-value

Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 < .001
14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 < .001
21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008
Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002
14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006
21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01
Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01
14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19
21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16
a-c
Means with different superscripts in each row are significantly different (P < 0.05).
Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point
1
NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P.
acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA,
butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.
2
P-Ch, Post-Challenge

Table 5. Effects of dietary treatments on the morphology of jejunum (μm) of broilers.

Treatments1
2
Items Day P-Ch NC PC PA MOS BA MA SEM P-value

Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36
14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 < .001
21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 < .001
Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73
14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41
21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74
VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01
14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002
21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008
a-c
Means with different superscripts in each row are significantly different (P < 0.05).
Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point
1
NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P.
acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA,
butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium.
2
P-Ch, Post-Challenge.

Challenging the birds with S. Typhimurium de-
creased FI, BWG, and feed efficiency, which is in
agreement with a number of former studies (Vande-
plas et al., 2009; Marcq et al., 2011). The reduced
performance observed in the challenged birds is prob-
ably due to intestinal mucosal damage induced by the
S. Typhimurium (Vandeplas et al., 2009). In contrast,
Mountzouris et al. (2009) and Amerah et al. (2012)
found that challenging birds with Salmonella had no
effect on performance response; this may be due to
discrepancies between the species, strains or dose of
Salmonella administered leading to different extents of
 SALMONELLA CHALLENGE AND FEED ADDITIVES
intestinal damage (Malago et al., 2003), or because of
differences in dietary cereal type fed to the birds (Teir-
lynck et al., 2009).
In the present study, dietary supplementation with
PA, BA, and MOS improved bird performance and
reduced the presence of Salmonella in the ceca. Inter-
estingly, only birds fed the mixture of additives (MA)
showed complete recovery following the challenge,
exhibiting similar BWG and FCR as the unchallenged
birds, highlighting a synergistic rather than being over-
lapping effect of the additives. Similarly, previous stud-
ies have reported that dietary supplementation with
prebiotic-based mannan-oligosaccharides (Lourenco
et al., 2016; Rajani et al., 2016), B. subtilis (Park and
Kim, 2014), and butyric acid (Saleem et al., 2016)
resulted in improved growth performance in broilers
challenged with Salmonella. The positive effect of
MOS on bird’s performance is likely attributed to
their ability to bind to pathogens, stimulating the
immune system and improving intestinal function by
enhancing villus uniformity (Baurhoo et al., 2007;
Lourenco et al., 2016; Pourabedin et al., 2016). Addi-
tionally, MOS-based prebiotics serve as a substrate for
endogenous beneficial bacteria, thus promoting com-
petitive exclusion of pathogenic microbes and selective
colonization of beneficial microbes (Spring et al., 2000;
Biggs et al., 2007). Fermentation of oligosaccharides
also results in production of short-chain fatty acids,
which display antimicrobial effects by penetrating
the cell membrane of gram negative bacteria, such as
Salmonella and coliforms, and altering the environ-
mental pH, leading to death of the bacteria (Faber
et al., 2012; Suiryanrayna and Ramana, 2015). The
beneficial effects of probiotic supplements on broiler
performance is associated with their role in maintaining
healthy balance of bacteria in the digestive tract and
improving metabolism and digestion by increasing
digestive enzyme activity (Sugiharto, 2016). Organic
acids potentially exhibit antimicrobial activities, by
reducing gastrointestinal tract pH and stimulating
protein digestion by converting pepsinogen to pepsin
(Suiryanrayna and Ramana, 2015). Thus, the observed
synergistic and complementary effect of the additives
used in this study is likely due to the dissimilar
beneficial mechanisms exhibited by each supplement.
The cecal samples of the unchallenged birds were
detected negative for the presence of Salmonella, in-
dicating that there was no cross contamination be-
tween the challenged and unchallenged birds. The di-
etary supplements used in the current study increased
Lactobacillus and Bifidobacterium concentration and
decreased the population of coliforms and Salmonella
in the ceca. In agreement with this study, Prado-
Rebolledo et al. (2017) and Pourabedin et al. (2016)
reported that supplementing LAB-based probiotics and
MOS-based prebiotics to the diets of broilers challenged
with Salmonella enterica decreased cecal colonization
of Salmonella. Similar effects on controlling and reduc-
2041
ing cecal Salmonella colonization have also been de-
scribed as a result of dietary application of organic acids
(Fernandez-Rubio et al., 2009; Saleem et al., 2016). The
metabolic activity of P. acidilactici bacteria results in
production of lactic acid and secretion of bacteriocins,
which create unfavorable conditions for the growth of
pathogenic bacteria such as coliforms and Salmonella
(Taheri et al., 2010). This suggests that the success of
the MA diet may be largely attributed to decreased
acid-binding capacity and consequential reduction in
digesta pH in birds fed this diet (Emami et al., 2017),
resulting in selective stimulation of beneficial bacteria,
increased production of short-chain fatty acids and thus
reduced colonization of pathogenic bacteria (Sugiharto,
2016).
The reliability of the H/L ratio as a biological index
of stress in avian species is well documented (Maxwell,
1993; Toghyani et al., 2011); heterophils display phago-
cytic, chemotactic, and adhesion activities and are re-
sponsible for protecting the body against pathogens
(Munyaka et al., 2012). The S. Typhimurium challenged
birds had significantly increased heterophil and reduced
lymphocyte numbers, causing the H/L ratio to increase
from 0.38 in the NC birds to 0.58 in the PC birds at d
21 post-challenge. The ratio decreased to approximately
0.46 in the challenged birds fed the diets supplemented
with PA, MOS, and BA. A complementary effect of the
additives was observed for the H/L ratio, with birds on
the MA treatment displaying similar ratios to those on
the NC treatment at d 14 and 21 post-challenge. These
findings are in agreement with previous reports high-
lighting that probiotics (Prado-Rebolledo et al., 2017),
prebiotics (Sadeghi et al., 2013) and short-chain organic
acids (Rath et al., 2006) possess immunomodulatory ef-
fects. LAB-based probiotics exert immunomodulatory
activities by interacting with the host immune system,
leading to changes in gene expression of cytokines and
thus altered intestinal microbiota composition (Men-
coni et al., 2011; Pourabedin et al., 2016). Janardhana
et al. (2009) demonstrated that, in addition to influenc-
ing the proliferative function and phenotypic expression
of immune cells, prebiotics can also influence systemic
antibody levels such as IgA, IgM, and IgG in broiler
chickens. Thus, it is probable that the systemic effect of
the supplements on immune response stimulation could
have alleviated the impact of the S. Typhimurium chal-
lenge on increasing the H/L ratio in the current study.
The S. Typhimurium challenge suppressed the activ-
ity of amylase, lipase and protease in this study. Some
species of pathogenic bacteria, such as Escherichia coli
(Zhang et al., 2016) and Clostridium (Mitsch et al.,
2004), have been shown to inhibit secretion of digestive
enzyme by damaging the villus and microvillus of the
intestinal mucosa. Dietary supplementation with PA,
MOS, BA, and MA ameliorated the negative impact of
the challenge on digestive enzyme activity, particularly
in the older birds. This is in agreement with a num-
ber of previous studies that have observed improved
2042 JAZI ET AL.
digestive enzyme activity in birds fed probiotics (Wang
and Gu, 2010; Zhang et al., 2016), and prebiotics (Xu
et al., 2003). The positive effect of these additives on
enzymes activity is likely attributed to their impact on
improving integrity of intestinal lining (Mitsch et al.,
2004) and modifying the microbial ecosystem; for ex-
ample, lactic-acid producing bacteria stimulate the se-
cretion of digestive enzymes and reduce gastrointestinal
pH (Jin et al., 2000; Xu et al., 2003; Suiryanrayna and
Ramana, 2015).
As stated by Choct (2009), the presence of stres-
sors throughout the intestinal tract can quickly alter
the structure of the intestine. Outcomes of the VH and
CD measurements in this study, as indices of intestinal
integrity and development, suggest that although in-
testinal development was not immediately affected by
the challenge (determined at d 7 post-challenge), the S.
Typhimurium challenge resulted in a noticeable reduc-
tion in VH at d 14 and 21 post-challenge, leading to a
decreased VH: CD ratios. In agreement with the cur-
rent findings, Rajani et al. (2016) also observed reduced
VH and VH: CD ratio in the small intestine of broilers
challenged with S. Typhimurium. Dietary supplemen-
tation with PA, MOS, BA, and MA improved VH and
VH: CD ratio, which is reflected by the growth per-
formance responses, in that shorter villi have a smaller
surface area for nutrient absorption. Previous studies
have also reported the beneficial effects of P. acidi-
lacticis, MOS and organic acids on intestinal morphol-
ogy (Baurhoo et al., 2007; Panda et al., 2009; Taheri
et al., 2010). According to the literature and as ob-
served in current study, evidently the improved intesti-
nal microbiota balance in favor of beneficial bacteria
by using the aforementioned additives can be respon-
sible for the rectified jejunum morphological parame-
ters (Antongiovanni et al., 2007; Panda et al., 2009;
Mallo et al., 2012).
CONCLUSION
The results obtained in the present study indicate
that S. Typhimurium challenge does not directly in-
fluence bird mortality, but it negatively affects growth
performance by impairing gut morphology, digestive en-
zyme secretion and microbiota composition, resulting in
reduced feed efficiency and bird health. The probiotic,
prebiotic and butyric acid supplements fed in this study
were, to some extent, efficacious at combatting the neg-
ative effects of the challenge on broiler performance and
gut health. However, a complete recovery from the chal-
lenge effects was observed only when a mixture of the
additives was administered, suggesting there was a syn-
ergistic effect between the additives. Therefore, it can
be concluded that dietary supplementation with a com-
bination of MOS, PA, and BA has the potential to be
considered as a practical and effective strategy for con-
trolling the incidence of Salmonella in broiler chickens
in a post-antibiotic era.
ACKNOWLEDGMENTS
The authors greatly acknowledge and appreciate the
technical support from the staff of the microbiology lab-
oratory, College of Medical Sciences, Islamic Azad Uni-
versity, Isfahan (Najafabad), Iran. We are also grateful
to Tak Genezist Company and Dr. Alireza Bidram for
supplying the probiotic and butyric acid.
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