Probiotics and Antimicrobial Proteins (2019) 11:981–989

https://doi.org/10.1007/s12602-018-9459-y

Effects of Dietary Supplementation of Probiotic Mix and Prebiotic

on Growth Performance, Cecal Microbiota Composition, and Protection
Against Escherichia coli O78 in Broiler Chickens
Reda Tarabees 1 & Khaled M. Gafar 2 & Mohamed S. EL-Sayed 1 & Awad A. Shehata 3,4 & Marwa Ahmed 5

Published online: 4 September 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
The current study conducted to investigate the effects of a multi-strain commercial probiotic mix and prebiotic
(isomaltooligosaccharide, IMO) on broiler performance parameters, cecal microbiota composition, and protection against challenge
with avian pathogenic Escherichia coli (APEC) O78. For this purpose, 101-day-old Cobb chicks were randomly allocated into four
experimental groups (G)—G01: basal diet, G02: basal diet and challenged with E. coli O78 at 28 days old, G03: basal diet with
probiotic mix and challenged with E. coli O78 at 28 days old, and G04: basal diet with IMO and challenged with E. coli O78 at 28 days
old. Results showed that weekly body weights in G03 were heavier (P < 0.05) than those of G01 and G02 at the fourth and fifth week.
The body gain at the fourth and fifth week was higher (P < 0.05) in G03 than those of the other groups. The hot carcass weight (g) was
significantly higher in broiler chickens kept in G03 and G04 compared with those in the control groups (G01 and G02). The probiotic
mix and IMO significantly increased the total lactobacilli and total lactobacilli-enterococci populations in the ceca of treated broilers,
respectively compared with those in the control groups. The treated broilers (G03 and G04) also showed lower mortality percentage
and E. coli recovery rates the liver and spleen than those in G02. It was concluded that probiotic mix or IMO significantly improved the
growth performance and modulated the intestinal microbiota of broiler chickens challenged with APEC O78.

Keywords Probiotic . Isomaltooligosaccharide . Broilers . Intestinal microbiota . Escherichia coli O78

Introduction related to diseases are regularly recurrent and results in severe

economic losses. For many decades, antibiotics have been used
The poultry industry has become an imperative pillar of the nation- as a single prevention scheme to combat such infections [1].
al income in many countries worldwide. Poultry meat offers an Excessive usage of antimicrobials as growth promoters in animal
appropriate substitute for many families of low income to compen- feed increased the number of multi-drug-resistant pathogens and
sate for the deficiency of other types of animal proteins. In large- turned these animals into potential reservoirs of antibiotic resis-
scalefarms,birdssubjectedtodifferenttypesofstressors;problems tance genes [2]. The consumption of antibiotic-contaminated food
products can demoralize the efficacy of antibiotic to combat bac-
terial infections, alter the gut microbiota balance, and increase the
* Reda Tarabees severity of chronic infections [3]. Therefore, many countries
[email protected] worldwide banned the use of antibiotics as growth promoters in
animal feed [4]. This is urging the scientific society to seek for
1
Department of Bacteriology, Mycology, and Immunology, Faculty of economic and efficient alternatives of high safety margins to re-
Veterinary Medicine, University of Sadat City, Sadat City, Egypt place in feed-antimicrobials [5]. Probiotics are nonpathogenic, live
2
Department of Nutrition and Clinical Nutrition, Faculty of Veterinary microorganisms, when supplied in adequate amounts exert a series
Medicine, University of Sadat City, Sadat City, Egypt of health benefits on the host [6]. Generally, in order to approve the
3
Department of Birds and Rabbit Diseases, Faculty of Veterinary usage of any bacteria as a probiotic candidate, it must be of host
Medicine, University of Sadat City, Sadat City, Egypt origin, resist the stomach acidity, and the bile salts in the intestine,
4
Faculty of Veterinary Medicine, Albrecht-Daniel-Thaer-Institute, able to adhere to gut mucosa, compete with other pathogens forgut
University of Leipzig, Leipzig, Germany colonization, and produce beneficial products that significantly
5
Directorate of Veterinary Medicine, El Hamoul, Minufyia, Egypt inhibited the growth of pathogenic bacteria [7–9]. Prebiotics are
982 Probiotics & Antimicro. Prot. (2019) 11:981–989

indigestible feed constituents that boost the host health by improv- Birds and Experimental Design
ing the growth and/or selectively enhance the activity of one or a
group of colon bacteria [10]. The application of probiotics and All procedures used in this 35-day experiment (5 weeks) were
prebiotics in broilers industry has been previously addressed in approved by the Animal Ethics Committee of Faculty of
many reports aimed to relieve stress, improve the immune re- Veterinary Medicine, University of Sadat City and complied
sponse [11, 12], and prevent the colonization of pathogenic bacte- with the Guidelines for the Care and Use of Animals in
ria [13]. Also, the use of both probiotics and prebiotics concurrent- Research. A total of 100 one-day-old commercial broiler
ly has been shown to modulate the intestinal microbiota composi- chicks (Cobb500) were randomly divided into four experi-
tion via increasing the number of beneficial bacteria in the colon of mental groups (25 each): G01) received a basal diet (negative
the treated poultry better than using each one individually [10, control), G02) received basal diet and challenged orally with
14–16]. 1 × 108 cfu/ml APEC O78 at 28 days old (positive control),
Escherichia coli is a typical resident of the chicken lower G03) received basal diet supplemented with the probiotic mix
gastrointestinal tract and also present in the upper respiratory (1 g/ kg) and challenged orally with 1 × 108 cfu/ml APEC
tract predominantly in the pharynx and trachea [17]. Although O78 at 28 days old, G04) fed basal diet with IMO supplement
the majority of E. coli serogroups are nonpathogenic, some (0.5 g/kg feed) and challenged orally with 1 × 108 cfu/ml
strains mainly avian pathogenic E. coli (APEC) are virulent APEC O78 at 28 days old. Feed and drinking water (free from
and produce diseases in birds [18]. APEC is incriminated as a antibiotics) were provided ad libitum. The four experimental
cause of many localized and generalized infections in poultry groups received the same amount of feed and the weekly feed
collectively known as colibacillosis, which results in remark- consumption was determined. Cleaning and feeding patterns
able economic losses in the poultry industry worldwide maintained to prevent the cross-contamination during the en-
[19–21]. The vaccination of birds against E. coli is not the tire experiment. The ingredients and the composition of the
optimal intervention since there are many serogroups involved starter and finisher rations as presented in Table 1. The birds
in the field outbreaks and vaccines only afford guard against received the basal diet during the first 2 weeks and then shifted
homogenous serogroups [22]. Consequently, the field treat- to the finisher ration for the last 3 weeks.
ment depends mainly on the antimicrobial agents; and this
results in the revival of the public and food safety authorities’ Broiler Performance Parameters
concerns [17]. Therefore, the purpose of this study is to eval-
uate the effects of multi-strain probiotic mix-based or prebiot- On a weekly basis, 5 birds from each group were randomly
ic (IMO)-based diets individually on the growth performance selected and weighed and the feed intake per each group was
parameters, cecal microbial profile, and protection against in- recorded at the same time. Feed intake was determined as the
fection with APEC O78 in commercial broiler chickens. difference between the amount of feed supplied and the re-
maining feed at the end of each week for each repetition. Body
weight (the weight of the birds at the end of each week) and
Materials and Methods body weight gain (the difference between the two successive
weights) were calculated. The feed conversion ratio (FCR)
Probiotic and Prebiotic was calculated as the ratio between feed intake and body
weight gain at the end of each week. The feed efficiency and
A multi-strain commercial probiotic mix in powder form con- the cumulative feed conversion ration were calculated. In ad-
taining Bacillus subtilis MORI 91 (2 × 108 colony forming dition, the relative growth rate percent (the percentage of body
unit (cfu)/g), Clostridium butyricum M7 (2.06 × 108 cfu/g), weight difference between two weights divided by the average
Lactobacillus plantarum K34 (2 × 108 cfu/g), functional or- of the same weights) was calculated weekly. At the end of the
ganic acid, soluble β-glucan, nucleotides, and experiment (35 days), five birds from each group were select-
deoxynojirimycin was used at a dose of 1 g/kg feed (Mori ed and the hot carcass weight (g) and the dressing percent
Max, UniTrade, Egypt). The prebiotic IMO (Jiangsu, China) were calculated.
was used at a dose of 0.5 g/kg feed. The probiotic mix and
IMO regularly supplemented in feed all over the whole exper- The Effect of Probiotic Mix or IMO Supplements
iment following the manufacturer instructions. on Cecal Microbiota Composition

Challenge Bacterium At 28 days old (4 weeks), five random birds were collected
from each group and slaughtered and the cecal contents (n = 5)
The APEC O78 kindly provided by the National Research were aseptically collected and kept at −20 °C for further bac-
Institute, Cairo, Egypt. APEC O78 (1 × 108 cfu/ml) was used teriological examinations. The cecal contents were serially
for the challenge at 28 days old. diluted in PBS for total aerobic, total anaerobic, enterococci,
Probiotics & Antimicro. Prot. (2019) 11:981–989 983

Table 1 The basal diets and their calculated nutrients composition (%) performed on Gassner agar (Oxoid, Germany) and the confir-
Ingredients Starter Grower- mation of the isolates was carried out using the MALDI-TOF
ration finisher profile according to the protocol previously established [23].
ration

Diet composition Statistical Analysis

Corn % 52.8 58.94
Soybean meal % 35.2 30 After completing the data collection, the data of the bacterial
Corn gluten % 6 5 counts were logarithmically transformed in order to obtain the
Soya oil % 2.6 2.57 normal distribution of data and the results representing as a
Common salt % 5.304 0.3 geometric mean [24]. The obtained data were enrolled into
Lysine % 5.1 0.1
statistical analysis via SPSS software (SPSS 15.0 Command
Methionine % 5.20 0.15
Syntax Reference. Chicago, IL: SPSS Inc). The analysis was
Di calcium phosphate % 1.6 1.7
done by applying a one-way analysis of variance together with
Duncan’s multiple range test for testing the significant differ-
Limestone % 1.2 1.118
ences among the different treatment groups. The results were
Broiler premix % 5.304 0.30
considered significant at P < 0.05.
Nutrient composition
ME Kcal/kg diet 1380.04 2846.9
Crude protein % 23.696 21.3065
Calcium % 0.9845 0.9625 Results
Available phosphorus % 0.4913 0.4984
Lysine % 1.1458 1.0116 Body Weight Development
Methionine % 0.4026 0.3665
The recorded effects of probiotic mix or IMO-based diets
supplements on the body weight development in broiler
total coliforms, and lactobacilli bacterial counts. The total aer- chickens challenged with APEC O78 are presented in
obic cell numbers were counted on sheep blood agar (Oxoid, Table 2. Results showed that, during the first and the fourth
Germany), total coliforms on MacConkey agar (Oxoid, week, the weekly body weight development in broiler
Germany) enterococci on citrate azide tween carbonate agar chickens received probiotic mix- or IMO-based diets (G03
(SIFIN, Berlin), total anaerobe on sheep blood agar (Oxoid, and G04) was significantly (P < 0.05) higher than those in
Germany), Lactobacillus on de Man, Rogosa, and Sharpe the control groups (G01 and G02). In contrast, there was no
Lactobacillus agar (SIFIN, Berlin), and Clostridium major change in the weekly body weight of broiler chickens
perfringens on sheep blood agar containing polymyxin B kept in these groups (G03 and G04, respectively) compared
and neomycin (Oxoid, Germany). The total aerobic cell num- with those in the control groups (G01 and G02) during the
ber, total coliforms number, and enterococci were cultured second and the third week. While, in the fifth week, only birds
aerobically at 37 °C for 24 h. The total anaerobic cell number fed with the probiotic mix as a dietary supplement (G03)
and lactobacilli were cultured at 37 °C for 48 h in anaerobic showed a significant (P < 0.05) higher weekly body weight
conditions. The full identification of the isolates was carried development (1850 g) than those in the other groups (G01,
out using the Matrix Assisted Laser Desorption/Ionization G02, and G04), as shown in Table 2.
Time of Flight (MALDI-TOF) profile according to the proto-
col previously established [23]. Body Gain

Response to Challenge At the first week, broilers received diets supplemented with a
probiotic mix or IMO (G03 and G04, respectively) exhibited a
The protective role of probiotic mix and the IMO- based feed significantly (P < 0.05) higher weekly body gain (g) com-
additives against challenge with E. coli O78 was assessed pared with those in the control groups (G01 and G02), as
based on the clinical signs, mortality percent, and the isolation given in Table 2. Conversely, during the second and the third
of E. coli from the visceral organs mainly liver and spleen at week, there was no significant difference between the groups
the seventh-day post challenge (35 days old). At the end of the receiving supplements (G03 and G04) and those receiving the
experiment (35 days old), five random birds from each group control diet (G01 and G02). While, in the fourth and the fifth
were slaughtered, and their liver (n = 5) and spleen (n = 5) week, birds fed the probiotic mix (G03) showed a significant-
were aseptically collected and stored at −20 °C for further ly (P < 0.05) higher weekly body gain (g) when compared to
bacteriological examination. Isolation of the E. coli was other treatments.
984 Probiotics & Antimicro. Prot. (2019) 11:981–989

Table 2 The effect of dietary supplementation of the probiotic mix and isomaltooligosaccharide (IMO) on the growth performance parameters of
broiler chickens

Parameter Age (weeks) G01 G02 G03 G04

Body weight (g) 1 170.00 ± 5.70b 168.00 ± 5.15b 190.00 ± 3.54a 194.00 ± 6.96a
2 507.00 ± 21.89a 497.00 ± 13.38a 532.00 ± 13.29a 533.00 ± 21.19a
3 914.00 ± 23.95a 922.00 ± 23.54a 958.00 ± 15.70a 933.00 ± 26.15a
4 1208.00 ± 38.91b 1216.00 ± 28.04b 1355.00 ± 17.89a 1318.00 ± 25.38a
5 1500.00 ± 50.00b 1500.00 ± 35.36b 1850.00 ± 35.36a 1521.00 ± 40.88b
Body gain (g) 1 120.00 ± 5.70 b 118.00 ± 5.15 b 140.00 ± 3.54 a 144.00 ± 6.96 a
2 337.00 ± 16.48 a 329.00 ± 8.28 a 342.00 ± 9.82 a 339.00 ± 15.76 a
3 407.00 ± 7.68 b 425.00 ± 13.23 ab 426.00 ± 5.79 ab 400.00 ± 5.48 b
4 294.00 ± 21.06b 294.00 ± 12.88b 397.00 ± 5.83a 285.00 ± 8.06b
5 292.00 ± 17.72 c 284.00 ± 9.27 c 495.00 ± 17.89 a 303.00 ± 18.55 bc
Relative growth rate (%) 1 108.85 ± 2.37bc 108.04 ± 2.25c 116.59 ± 1.23a 117.78 ± 2.22a
2 99.43 ± 1.12a 98.98 ± 0.36a 94.69 ± 0.59b 93.18 ± 1.60b
3 57.48 ± 1.93ab 59.91 ± 1.19a 57.24 ± 1.03ab 54.71 ± 1.11b
4 27.63 ± 1.48b 27.53 ± 1.14b 34.35 ± 0.57a 26.57 ± 1.03b
5 21.55 ± 0.98b 20.91 ± 0.43b 30.85 ± 0.62a 22.06 ± 0.96b
Feed efficiency% 1 0.38 ± 0.02a 0.37 ± 0.02a 0.35 ± 0.01a 0.40 ± 0.02a
2 0.84 ± 0.04 a 0.82 ± 0.02 a 0.71 ± 0.02 b 0.77 ± 0.04 ab
3 0.68 ± 0.01ab 0.71 ± 0.02 a 0.63 ± 0.01 c 0.63 ± 0.01c
4 0.39 ± 0.03 a 0.39 ± 0.02 a 0.43 ± 0.01 a 0.36 ± 0.01 a
5 0.37 ± 0.02 b 0.36 ± 0.01 b 0.52 ± 0.02 a 0.33 ± 0.02 b
FCR 1 2.69 ± 0.13a 2.73 ± 0.13a 2.86 ± 0.07a 2.52 ± 0.11a
2 1.20 + ±0.06b 1.22 ± 0.03b 1.41 ± 0.04a 1.31 ± 0.06ab
3 1.48 ± 0.03b 1.42 ± 0.05b 1.60 ± 0.02a 1.60 ± 0.02a
4 2.64 ± 0.18ab 2.61 ± 0.12ab 2.32 ± 0.03b 2.82 ± 0.08a
5 2.78 ± 0.16b 2.83 ± 0.10b 1.95 ± 0.07a 2.08 ± 0.20a
Cumulative FCR 1.93 ± 0.06ab 1.92 ± 0.05ab 1.86 ± 0.04b 2.08 ± 0.06a

FCR feed conversion ratio

G01: Negative control, fed a basal diet, G02: positive control treatment, fed a basal diet and challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old,
G03: fed a basal diet containing probiotic mix (1 g/kg feed) and challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old, G04: fed a basal diet
containing IMO (0.5 g/kg feed) and challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old
a-b
Means (n = 5) within a row with different superscripts differ significantly (P ˂ 0.05)

Relative Growth Rate Feed Efficiency

The effect of probiotic mix or IMO supplementations on the The data presented in Table 2 showed that there was no
relative growth rate (%) is presented in Table 2. During the first- significant difference in the relative growth rate (%) be-
week broiler chickens receiving diets containing probiotic mix tween broilers receiving the probiotic mix or IMO supple-
or IMO (G03 and G04 respectively) recorded a significantly mented diets (G03 and G04) and those receiving the con-
(P < 0.05) higher relative growth rate (%) when compared with trol diet (G01 and G02) during the first and the fourth
those receiving the basal diet (G01–G02). In contrast, during week. While, in the third week, broiler chickens receiving
the third week, broilers chickens kept in G03 and G04 showed a the probiotic mix or IMO supplemented diets (G03and
significantly lower relative growth rate when compared with G04) showed a significant lower feed efficiency (%) com-
those kept in the control groups G01 and G02. On the other pared with those in the control groups (G01and G02). On
hand, in the fourth and fifth week, only birds fed a basal diet the contrary, in the fifth week, only broilers kept in G03
with the probiotic mix (G03) showed a significantly higher (basal diets + probiotic mix) exhibited a higher weekly
weekly relative growth rate (%) when compared with those in feed efficiency rate (0.52 ± 0.02) than those kept in the
the other groups (G01, G02, and G04) (P < 0.05). other groups (P < 0.05).
Probiotics & Antimicro. Prot. (2019) 11:981–989 985

Feed Conversion Ratio Table 3 The effect of dietary supplementation of probiotic mix and
IMO on broiler carcass quality parameters (hot carcass weight (g) and
dressing %)
Data presented in Table 2 showed that there was no significant
variation in the feed conversion ratio between broiler chickens Groups Hot carcass weight Dressing %
in groups receiving the probiotic mix or IMO supplemented
G01 1170.00 ± 37.42b 78.04 ± 0.99b
diets (G03 and G04) and those receiving the basal diet (G01
G02 1160.00 ± 50.99b 77.19 ± 1.72b
and G02) in the first week. In contrast, during the second
G03 1410.00 ± 40.00a 76.17 ± 0.82b
week, broilers fed with the probiotic mix showed a significant
G04 1350.00 ± 44.72a 88.73 ± 1.21a
higher FCR when compared with those in the control groups
(G01 and G02). The data also showed that during the third- G01: Negative control, fed a basal diet, G02: positive control treatment,
week broilers fed with a probiotic mix or IMO supplemented fed a basal diet and challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-
diets showed a significant higher FCR than those in the con- old, G03: fed a basal diet containing probiotic mix (1 g/kg feed) and
challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old, G04: fed a
trol groups (G01 and G02). While, in the fourth week, only
basal diet containing IMO (0.5 g/kg feed) and challenged with 1 ×
broilers fed diet supplemented with the probiotic mix exhibit- 108 cfu/ml E. coli O78 at 28-day-old
ed significantly lower FCR (P < 0.05) compared with those a-b
Means (n = 5) with different superscripts within a column differ sig-
receiving diet supplemented with IMO (G04). In the fifth nificantly (P ˂ 0.05)
week, broilers fed probiotic mix or IMO supplements in the
diet (G03 and G04) showed significantly lower FCR (P < Protective Effect of Probiotic Mix and IMO
0.05) compared with those in the control groups (G01 and against Challenge with APEC O78
G02). Concerning the cumulative FCR, broiler chickens in
G03 exhibited lower cumulative FCR when compared with Broilers kept in G01 showed no clinical signs. While, those
those in the other groups (G01, G02, and G04), as presented in kept in G02 (fed a basal diet and challenged with E. coli
Table 2. The data also showed that broilers kept in G03 O78 at 28 days old) showed depression, diarrhea, and respi-
showed a significantly lower cumulative FCR (1.86 ± 0.04) ratory manifestations at a 3-day post challenge. The main
when compared with those in G04 (Table 2). postmortem lesions were septicemia, congested liver and
spleen, enlarged kidneys, air sacculitis, and pericarditis.
Hot Carcass Weight and Dressing Percent Finally, the probiotic mix- and the IMO-supplemented broiler
chickens (G03 and G04) showed moderate clinical signs in-
The data presented in Table 3 showed that broilers receiving cluding depression and diarrhea with no clear lesions in the
diets supplemented with a probiotic mix or IMO (G03 and visceral organs (liver and spleen). The data presented in
G04) recorded notably (P < 0.05) higher hot carcass weight Table 5 showed that the mortality percent were 0%, 13.6%,
(g) compared with those receiving basal diet (G01 and G02), 0%, and 5% of birds kept in G01, G02, G03, and G04, respec-
as presented in Table 3. Concerning the dressing percent, tively. Moreover, E. coli was isolated from 4/5 (80%) and 5/5
broilers receiving IMO-supplemented diet (G04) recorded a (100%) of liver and spleen samples collected from the positive
significantly (P < 0.05) higher dressing percent when com- control (G02), respectively. While in the case of probiotic
pared to those in other groups (G01, G02, and G03), as pre- mix-supplemented broilers (G03), E. coli was isolated from
sented in Table 3. one liver sample (20%). In contrast, in the case of broilers
receiving IMO supplement in the diet (G04), E. coli was re-
Modulatory Effect of Probiotics and Prebiotic on Cecal covered from 1/5 (20%) and 2/5 (40%) of liver and spleen
Microbiota Composition samples, correspondingly. Finally, E.coli was unsuccessfully
recovered from the visceral organs of broilers kept in G01
The effect of dietary supplementation of probiotic mix or IMO (negative control), as shown in Table 5.
on the cecal microbiota composition is presented in Table 4.
Chickens fed with a probiotic mix or IMO-based diets (G03
and G04) exhibited higher (P < 0.05) cecal lactobacilli popu-
lation compared with those in the control groups (G01 and Discussion
G02). While birds fed a basal diet with IMO (G04) showed
a significantly higher enterococci population than the birds The study herein investigated the effects of Bacillus subtilis
kept in other groups (G01, G02, and G03). In contrast, there MORI 91, Clostridium butyricum, Lactobacillus plantarum
is no significant change in the number of the total aerobes, K34 based-probiotic mix or IMO-based prebiotic as feed sup-
total anaerobes, total coliforms, and total Clostridium plements individually on the broilers performance parameters,
perferingens in the ceca of the broiler chickens in all groups, cecal microbiota balance and the protection against visceral
as shown in Table 4. invasion with APEC O78. The obtained results showed that
986 Probiotics & Antimicro. Prot. (2019) 11:981–989

Table 4 The modulatory effect of

probiotic mix and IMO on the Parameter G01 G02 G03 G04
cecal microbiota composition
(cfu/g) of commercial broiler Total bacterial count (aerobes) 9.5 ± 0.14a 9.1 ± 0.33a 9.4 ± 0.43a 9.4 ± 0.65a
a
chickens at 28-day-old Total bacterial count (anaerobes) 9.3 ± 0.23 9.6 ± 0.21a 9.7 ± 0.89a 8.9 ± 0.22a
Enterococci 5.6 ± 0.14b 5.8 ± 0.23b 5.4 ± 0.91b 6.2 ± 0.12a
Total coliforms 6.43 ± 0.66a 6.31 ± 0.71a 5.6 ± 0.12a 5.8 ± 0.11a
Lactobacilli 7.3 ± 0.18b 7.4 ± 0.14b 8.6 ± 0.13a 8.2 ± 0.11a
C. perfringens 6.7 ± 0.32a 6.2 ± 0.1a 6.5 ± 0.37a 6.7 ± 0.15a

G01: Negative control, fed a basal diet, G02: positive control treatment, fed a basal diet and challenged with 1 ×
108 cfu/ml E. coli O78 at 28-day-old, G03: fed a basal diet containing probiotic mix (1 g/kg feed) and challenged
with 1 × 108 cfu/ml E. coli O78 at 28-day-old, G04: fed a basal diet containing IMO (0.5 g/kg feed) and
challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old
a-b
Means (n = 5) with different superscripts within a row differ significantly (P ˂ 0.05)

dietary supplementation with 2 × 108 cfu/ml, 2.06 × 108 cfu/ significant improvement in growth performance parameters
ml, and 2 × 10 8 cfu/ml of Bacillus subtilis MORI 91, and cumulative CFR compared with those in the other groups
Clostridium butyricum, Lactobacillus plantarum K34 as a (G01, G02, and G04). This effect could be a result of in-
probiotic mix-based or IMO as prebiotic-based diets could creased activities of digestive enzymes including amylase
be the optimal intervention to maintain high growth perfor- and protease [27] and other factors were not investigated un-
mance in broiler chickens challenged with APEC O78. In der the conditions of the current study. These findings are
addition, it could reduce the emergence of antibiotic-residues consistent with many studies that verified the positive effects
in fresh and frozen poultry by-products [25, 26]. Lactobacillus of the probiotics on broilers growth performance [32–34].
strains have been widely used as a probiotic candidate in Previous reports showed that diets supplemented with a
large-scale farms to enhance the growth performance param- single-strain probiotic [35] or multi-strain probiotic [36] sig-
eters, increase the carcass yield, and improve the carcass qual- nificantly improved the growth performance in the treated
ity [27]. Clostridium butyricum, a butyric acid-producing an- broilers comparing with those in the control treatment.
aerobic microorganism commonly found in soil and in the gut Huang et al. reported that broilers received diets with 332,
of healthy animals and humans [28]. Clostridium butyricum 425 mg/kg of Lactobacillus acidophilus exhibited a signifi-
has been used as feed additive in poultry diets to improve the cantly higher body weight and body weight gain compared
growth performance, augment the immune system, adjust the with those of the control treatments [37]. Zhang et al. revealed
intestinal microbial balance and composition, and protract the that dietary supplementation with 105 and 108 cfu/kg of a
intestinal morphology [29–31]. In the present study, broilers Bacillus-based probiotic improved the body weight gain in
receiving diet supplemented with Bacillus subtilis MORI 91 the treated broilers compared to the controls [38, 39]. Yang
(2 × 108 cfu/g), Clostridium butyricum M7 (2.06 × 108 cfu/g), et al. found that broilers fed a diet with 2 × 107 or 3 × 107 cfu/
and Lactobacillus plantarum K34 (2 × 108 cfu/g) showed kg of C. butyricum showed improved average daily gain com-
pared to those of the controls [40]. In contrast, others have
Table 5 Response of broiler chickens toward challenge with E. coli stated that probiotics had no positive effect on broiler perfor-
O78 in terms of mortality % and E. coli recovery from the visceral mance [41, 42]. The data presented also stated that a signifi-
organs (liver and spleen)
cant improvement in the cumulative FCR was observed in
Group no. Mortality E. coli recovery from internal organs broilers given diet containing probiotic mix supplement com-
pared with those in the other treatment groups. Improved cu-
No % Liver Spleen mulative FCR is desirable as it reflects the efficient utilization
G01 0 0 0/5 (0%) 0/5 (0%)
of the nutrients. This outcome is in line with the finding of
Abudabos et al. who reported significant improvement in cu-
G02 3 13.6 4/5 (80%) 5/5 (100%)
mulative FCR of broilers received probiotic supplements
G03 0 0 0/5 (0%) 1/5 (20%)
compared to those in the control groups [43]. Conversely,
G04 1 5 1/5 (20%) 2/5 (40%)
Mountzouris et al. did not observe any significant change in
G01: Negative control, fed a basal diet, G02: positive control treatment, the cumulative FCR between the birds in treated and untreated
fed a basal diet and challenged with 1 × 108 cfu/ml E. coli O78 at 28-day- groups [44]. The inconsistency in the effects of probiotics be-
old, G03: fed a basal diet containing probiotic mix (1 g/kg feed) and tween these studies and our study could be due to the compo-
challenged with 1 × 108 cfu/ml E. coli O78 at 28-day-old, G04: fed a
basal diet containing IMO (05 g/kg feed) and challenged with 1 × sition and the type of the probiotic, the way of administration,
108 cfu/ml E. coli O78 at 28-day-old the dose of the probiotic, and the breed of the broiler [45, 46].
Probiotics & Antimicro. Prot. (2019) 11:981–989 987

Prebiotics mainly oligosaccharides have been expansively challenged with APEC O78 compared with positive and nega-
used as a feed additive in the broiler industry; it encourages the tive control groups (G01 and G02). This outcome is not in agree-
growth and multiplication of beneficial gut microflora and ment with the finding of Thitaram et al. [51], who stated that 1%
boosts the immune system of chickens [47, 48]. In the present IMO supplement had no significant effect on the number of the
study, the relative growth rate (during the first 2 weeks), feed beneficial bacteria in the colon of treated birds compared with the
efficiency, and the dressing percent were significantly im- controls. IMO is an indigestible oligosaccharide when a part
proved in broilers fed diet supplemented with IMO (G04) reached the colon, it easily utilized as a source of carbohydrates
compared with those in the other treatment groups. These by the beneficial bacteria including lactic acid bacteria [48].
findings are in accordance with that obtained by Nikpiran Increased number of lactic acid bacteria in the colon creates
et al. who reported that broiler chickens receiving diet- unfavorable conditions for the growth of pathogenic bacteria
containing oligosaccharides showed a significantly higher [51]. Increased number of lactobacilli in the ceca of broiler
body weight than those in the other groups [49]. In contrast, chickens resulted in the improvement of the intestinal function,
Appelt et al. did not find any major variation in the weight maturation of the gut microflora, enhancement of the immune
gain between broilers fed probiotics and prebiotic- response [2], prevention of the colonization, and shedding of
supplemented diets and those fed basal diets [50]. Thitaram many enteric pathogens such as Campylobacter jejuni [56] and
et al. stated that poultry received diets containing 2% or 4% Salmonella Enteritidis [57], and reduction of the broiler mortality
IMO achieved similar body weight to those in the control rates [58]. In the present study, the mortality percentages and the
groups [51]. The discrepancy in results between study herein recovery rates of E. coli from the visceral organs of broilers
and the previous studies can be attributed to many factors receiving a probiotic mix or IMO-based supplements (G03 and
including, the housing conditions, the composition of the di- G04) and challenged with APEC E. coli O78 were lower when
ets, the dose, and the type of the additive, route of the admin- compared with those kept in the positive control group (G02).
istration, and the breed of the broiler [51]. These results are in line with many reports that showed, dietary
In poultry farms, maintaining a healthy equilibrium of micro- supplementation of C. butyricum boost the immune response,
organisms in the gastrointestinal tract of broilers is important for enhanced the intestinal barrier function, and significantly
optimal animal well-being and reproduction. Probiotics and pre- inhibited the E. coli-induced intestinal disorders of broiler
biotics help to maintain and adjust the intestinal microbiota com- chickens challenged with E. coli K88 [59, 60]. There are many
position which considerably prevents the colonization of patho- studies described possible mechanisms of probiotics/prebiotics/
genic bacteria [52–54]. In the present study, there is no significant and pathogen invasion. Dietary supplementation of probiotics-
difference in the total bacterial counts between the groups fed based diets significantly reduced the pathogen visceral invasion,
with the probiotic mix or IMO supplemented diets and the it can be a result of two main mechanisms: by exclusion as well
groups fed the basal diet. These outcomes are consistent with as by competition [61]. While in the case of prebiotic-based diets,
the findings of Mountzouris et al. who reported that there was a proportion of the IMO reached the intestine undigested and acts
no significant difference in the total coliform and total aerobic as a carbohydrate substrate that easily utilized by the beneficial
counts between broilers received probiotics-based diets and those flora in the ceca and significantly modified the intestinal micro-
in the control groups [44]. In contrast, the cecal lactobacilli and bial composition [51]. Increased number of the beneficial flora
the cecal lactobacilli-enterococci populations tended to increase such as enterococci and lactobacilli significantly altered pathogen
significantly in groups supplemented with probiotic mix and visceral invasion in a competitive exclusion manner [48, 51]. In
IMO-based diets (G03 and G04), respectively compared to the the current study, the probiotic mix-based feed additive tended to
groups received basal diet (G01 and G02). These findings are increase the number of the lactobacilli in the ceca of the treated
consistent with those of Mountzouris et al. [44], who reported group (G03) on day 28 compared with the control treatment,
that probiotics significantly increased the lactobacilli populations which reflected positively on the growth performance parameters
in the intestine of the treated broilers compared with those in the of birds. This is inconsistent with the findings of Olnood and co-
control groups. Conversely, Giannenas et al. [55] did not report authors, who did not demonstrate any significant improvement in
any significant increase in the number of lactobacilli and entero- the growth performance of birds received probiotics supplements
cocci in the cecal microbiota of probiotics or prebiotics- and have a higher number of lactobacilli in their ceca compared
supplemented broilers compared with those in the control with those fed non-supplemented diets [60]. This variation in
groups. Clostridium butyricum produces large amounts of acetic effects of probiotics can be due to the strains and the concentra-
acid, valeric acid, butyric acid, and short-chain fatty acids, which tion of the probiotic bacteria used as supplements, the housing
significantly enhanced the growth of the beneficial bacteria in the conditions, and the breed of broiler [61]. In conclusion, under the
colon and banned the growth and colonization of other patho- conditions of the current study, a mixture of Bacillus subtilis
genic bacteria [31]. In the theme, IMO in a dose of 0.5 g/kg feed MORI 91 (2 × 108 cfu/kg), Clostridium butyricum M7 (2.06 ×
(G04) significantly increased the number of the lactobacilli and 108 cfu/kg), Lactobacillus plantarum K34 (2 × 108 cfu/kg)—
enterococci populations in the ceca of broiler chickens probiotic-based or IMO (0.5 g/kg feed) prebiotic-based feed
988 Probiotics & Antimicro. Prot. (2019) 11:981–989

additives individually could improve the growth performance definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol
14:491–502
parameters, modulate the cecal microbiota composition, and pro-
11. Donalson LM, McReynolds JL, Kim WK, Chalova VI, Woodward
tect against visceral invasion of APCE O78 in commercial CL, Kubena LF et al (2008) The influence of a fructooligosaccha-
broilers. In addition, these findings will open up avenues for ride prebiotic combined with alfalfa molt diets on the gastrointesti-
future research in the field of probiotics to understand how pos- nal tract fermentation, Salmonella enteritidis infection and intestinal
shedding in laying hens. Poult Sci 87(7):1253–1262
itively adjusted cecal microbiota composition affects the growth
12. Revolledo L, Ferreira AJP, Mead GC (2006) Prospects in
performance in broiler chickens challenged with APEC O78. Salmonella control: competitive exclusion, probiotics, and en-
hancement of avian intestinal immunity. J Appl Poult Res 15:
Funding Information The authors would like to acknowledge the Science 341–351
and Technology Development Fund, Egypt, Grant No. 9222 for their 13. Cummings JH, Macfarlane GT, Englyst HN (2001) Prebiotic diges-
partial support. tion and fermentation. Am J Clin Nutr 73:415S–420S
14. Awad WA, Ghareeb K, Abdel-Raheem S, Bohm J (2009) Effects of
dietary inclusion of probiotic and synbiotic on growth performance,
Compliance with Ethical Standards organ weights, and intestinal histomorphology of broiler chickens.
Poult Sci 88:49–56
Conflict of Interest The authors declare that there is no conflict of 15. Abdelqader A, Al-Fataftah AR, Das G (2013) Effects of dietary
interest. Bacillus subtilis and inulin supplementation on performance, egg-
shell quality, intestinal morphology and microflora composition of
Statement The authors would like to state that the manuscript has not laying hens in the late phase of production. Anim Feed Sci Technol
been published and is not under consideration for publication elsewhere. 179:103–111
16. Roul G, Panda N, Mishra PK, Behera PC, Panigrahi B, Pati PK
(2015) Effect of supplementation of probiotics and prebiotics on
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