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Effects of Lactobacillus plantarum on production performance, immune characteristics, antioxidant status, and intestinal microflora of bursin-immunized broilers Xuejiao Shen, Dan Yi, Xueqin Ni, Dong Zeng, Bo Jing, Mingxia Lei, Zhengrong Bian, Yan Zeng, Tao Li, and Jinge Xin

Abstract: Examples of probiotics that can promote host health by improving its intestinal microbial balance and intestinal immunity belong to the genus Lactobacillus. Bursin (BS) is a peptide isolated from the bursa of Fabricius for use as an adjuvant for a variety of immunogens. To investigate the synergistic effects of Lactobacillus plantarum (LP) dietary supplementation and BS immunization on production performance, immune characteristics, antioxidant status, and intestinal microflora in broilers, we randomly allocated 200 1-day-old broilers of mixed sex into 4 treatments in a 2 × 2 factorial arrangement (LP–/BS–, LP–/BS+, LP+/BS–, LP+/BS+) for 42 days. BS immunization enhanced immune response by increasing serum total immunoglobulin G concentration and interleukin-6 concentration, promoted antioxidant capacity by increasing catalase activities in serum and liver and by decreasing serum malondialdehyde (MDA) content at 42 days of age (DOA), and enriched intestinal microflora diversity. LP supplementation enhanced immune response by increasing interleukin-2 concentration at 42 DOA; promoted antioxidant capacity by increasing liver catalase activities, increasing glutathione peroxidase activities in serum and liver at 21 DOA, and decreasing serum MDA content at 42 DOA; promoted intestinal microflora composition by decreasing total aerobes and Escherichia coli counts at 21 DOA, by increasing total anaerobes count at 21 DOA, and by increasing Lactobacillus spp. and Bifidobacterium spp. counts at both 21 and 42 DOA. The interactions between BS and LP had a significant effect on daily body mass gain and feed conversion ratio in the starter period (1–21 DOA); on interleukin-2 concentration and liver MDA content at 21 DOA; and on thymus index, peripheral lymphocyte proliferation, and E. coli counts at 42 DOA. Overall, these data suggest that the combination of LP dietary supplementation and BS immunization promoted the production performance, immune characteristics, antioxidant status, and intestinal microflora of broilers. Key words: broiler, Lactobacillus plantarum, bursin, production performance, immune characteristic, antioxidant status, intestinal microflora. Résumé : Certaines bactéries du genre Lactobacillus peuvent favoriser la santé de l’hôte en améliorant son équilibre et son immunité intestinale. La bursine (BS) est un peptide issu de la bourse de Fabricius (BF) qui exerce une activité adjuvante a` l’endroit de plusieurs immunogènes. Afin d’étudier les effets synergiques d’un supplément alimentaire de Lactobacillus plantarum (LP) adjoint a` une immunisation avec BS sur la productivité, les caractéristiques immunitaires, l’état antioxydant et la microflore intestinale chez les poulets, 200 poussins d’un jour, de sexes mélangés, ont été répartis aléatoirement dans quatre groupes de traitements selon un arrangement factoriel de 2 × 2 (LP–/BS–, LP–/BS+, LP+/BS–, LP+/BS+) pendant 42 jours. L’immunisation avec la BS a intensifié la réponse immunitaire en haussant les concentrations sériques d’IgG total et d’IL-6, a favorisé la capacité antioxydante en rehaussant l’activité CAT sérique et hépatique, a diminué le contenu sérique en MDA a` 42 jours après l’éclosion (JAE), et a enrichi la diversité microbienne intestinale. La supplémentation avec LP a rehaussé la réponse immunitaire en augmentant la concentration d’IL-2 a` 42 JAE, a favorisé la capacité antioxydante en rehaussant l’activité CAT hépatique, a rehaussé l’activité GSH-Px sérique et hépatique a` 21 JAE, a diminué le contenu sérique en MDA a` 42 JAE; le probiotique a par ailleurs amélioré la composition de la microflore intestinale en diminuant les comptes totaux d’aérobies et d’Escherichia coli a` 21 JAE, et en faisant grimper le nombre total d’anaérobies a` 21 JAE et les comptes de Lactobacillus spp. et Bifidobacterium spp. a` 21 et 42 JAE. Il y eut des interactions notables entre la BS et LP au regard du gain de masse corporelle (GMC) quotidien et du taux de conversion de la moulée (TCM) dans la période du début (1 a` 21 JAE), de la concentration d’IL-2 et du contenu de MDA hépatique a` 21 JAE, de l’indice thymique (IT), de la prolifération des lymphocytes périphériques et des comptes d’E. coli a` 42 JAE. Dans l’ensemble, ces données tendent a` démontrer que l’association d’un supplément alimentaire de LP et de l’immunisation avec de la BS favorise la productivité, les caractéristiques immunitaires, l’état antioxydant et la microflore intestinale des poulets. [Traduit par la Rédaction] Mots-clés : poulets, Lactobacillus plantarum, bursine, productivité, caractéristiques immunitaires, état antioxydant, microflore intestinale.

Received 24 September 2013. Revision received 27 January 2014. Accepted 27 January 2014. X. Shen, D. Yi, X. Ni, D. Zeng, B. Jing, M. Lei, Z. Bian, Y. Zeng, T. Li, and J. Xin. Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Xinkang 46, Ya’an 625014, People's Republic of China. Corresponding author: Xueqin Ni (e-mail: [email protected]). Can. J. Microbiol. 60: 193–202 (2014) dx.doi.org/10.1139/cjm-2013-0680

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Introduction Probiotics, defined as “live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” by Fuller (1989), enhance production performance and immune responses of farm animals and poultry by modulating intestinal microbial balance and developing immunity of the intestinal tract. Because of the ban of subtherapeutic antibiotic usage in Europe, probiotics, classified as zootechnical feed additives, are considered to be a good alternative to the use of antibiotics. So far, a variety of probiotics have been used in poultry. Lactobacillus strains, the most abundantly used probiotic strains, have been found to be long-term residents of the intestinal tracts and have been determined to be beneficial additives for promoting poultry production performance (Jin et al. 1998b; Huang et al. 2004; Willis and Reid 2008), enhancing immune responses (Koenen et al. 2004; Stringfellow et al. 2011), improving antioxidant capacity (Wang et al. 2009; Zhang et al. 2010; Kang et al. 2012; Li et al. 2012), regulating the intestinal microflora composition (Jin et al. 1998b; Fuentes et al. 2008; Yu et al. 2008), and promoting the host health. Contemporary biosecurity threats arising from the increasing resistance of pathogens to antibiotics and the accumulation of antibiotic residues in animal products and the environment (Barton 2000) have ignited a call for a ban of worldwide subtherapeutic antibiotic usage. To reduce antibiotic usage, a combination of intervention strategies that aim to enhance the immune system of the host, such as immunization of potent adjuvants and applications of suitable feed and water additives, is worth trying. A wide variety of studies have reported on the addition of a single Lactobacillus strain to the diet. Recent evidence has suggested that probiotics may enhance host defenses and improve vaccine response (Koenen et al. 2004; Stringfellow et al. 2011). Since allochthonous strains elicit a substantial host response and since autochthonous strains are highly compatible with the intestinal environment of a host (Berg 1983), the administration of highly concentrated chicken-specific bacterial cultures isolated from a host, containing both adequate quantities of live beneficial microorganisms and their products of fermentation, is an effective way to benefit the host’s production performance and intestinal microflora (Jin et al. 1998b; Willis and Reid 2008). As well, the Lys-HisGly-NH2 tripeptide bursin (BS), the first peptide with a clear chemical structure, was isolated from the bursa of Fabricius (BF) in 1986 (Audhya et al. 1986). It has been shown to induce B lymphocyte proliferation and differentiation (Audhya et al. 1986; Yuko et al. 2001), stimulate stem cell differentiation (Lassila et al. 1989; Audhya et al. 1990), and promote immunoglobulin switching from immunoglobulin M (IgM) to IgG (Baba and Kita 1977). Furthermore, Wang et al. (2008a) concluded that BS may serve as a potent adjuvant for vaccine application. However, it is still unknown if the combined application of LP supplementation and BS immunization has synergistic effects. To address this issue, this study was designed to assess the synergistic effects of LP dietary supplementation on production performance, immune characteristics, antioxidant status, and intestinal microflora of BS-immunized broilers.

Materials and methods Lactobacillus plantarum culture preparation A strain of Lactobacillus previously isolated from the intestinal mucosa of healthy adult chicken was provided by the Animal Microecological Research Center (College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China). It was characterized by tolerance to bile acid and low pH, rapid growth rates, and high lactate production (Ding 2003). The strain was tentatively identified as Lactobacillus plantarum (LP) by 16S rDNA sequencing analysis and was maintained at –70 °C in de Man – Rogosa – Sharpe (MRS) broth supplemented with 20% (v/v) glycerol. Lastly,

Can. J. Microbiol. Vol. 60, 2014

the strain was transferred at least 3 times consecutively using 1% (v/v) inoculum in MRS broth at 37 °C for 24 h before use. Then, the LP culture was centrifuged at 2000g for 20 min at 4 °C and resuspended in MRS broth to obtain 1 × 109 colony-forming units (cfu)/mL. LP culture was mixed into the basal diet at a level of 1 g/kg (0.1%, m/m) each day to ensure viable bacterial cells in the feed throughout the experimental period. This level of supplementation has been shown to have positive effects on the production performance, serum lipid, and intestinal microflora of broilers (Jin et al. 1998b). The working culture was prepared weekly and then maintained at –20 °C in the MRS broth. Experimental design A total of 200 1-day-old broilers (Cobb500) of mixed sex with similar body mass (45.70 ± 0.35 g) were obtained from a local commercial hatchery. Broilers were randomly divided into 4 treatments (5 replicates per treatment). Each replicate was assigned to a floor pen (10 birds per pen). Treatments received a corn–soybean basal diet in mashed form. Experimental diets were formulated to meet the nutrient requirements recommended by the National Research Council (1994). The composition of the basal diet is shown in Table 1. The treatments in a 2 × 2 factorial arrangement were as follows: Treatment A was the control, L. plantarum nonsupplemented and bursin nonimmunized (LP–/BS–); Treatment B was L. plantarum nonsupplemented and bursin immunized (LP–/BS+); Treatment C was L. plantarum supplemented and bursin nonimmunized (LP+/BS–); Treatment D was L. plantarum supplemented and bursin immunized (LP+/BS+). LP supplementation was administered with the solution of LP culture at dose of 1 g/kg feed (0.1%, m/m) and water mixed into the basal diet per serving. BS (95.0% purity, Shanghai Shenlian Biotechnology Co., Shanghai, China) immunization was administered via intramuscular injection at 4 and 7 days of age (DOA) with 0.01 mg BS/kg body mass. Isometric sterile physiological saline was used as a placebo in those treatments not receiving LP or BS. Throughout, broilers in all pens had free access to feed and water. All broilers were maintained under a uniform environment with heat lamps to maintain an optimal temperature. The feeding trial was conducted in a poultry house of Sichuan Agricultural University for 42 days. Body mass of each pen was recorded on the morning of 21 DOA for the starter period (1–21 DOA) and 42 DOA for the grower period (22–42 DOA). Daily body mass gain (BMG) was calculated as the difference between the final and initial chicken masses. Feed intake (FI) per pen was recorded every day to determine daily FI. Feed conversion ratio (FCR) was calculated as the ratio between daily FI and daily BMG during each phase of the experimental period. In addition, overall daily BMG, daily FI, and FCR were calculated for the whole period of the experiment (1–42 DOA). All experimental procedures were performed following the recommendations of the institutional Animal Care and Use Committee of Sichuan Agricultural University. Sample collection Ten broilers per treatment (2 birds per pen) were randomly selected, weighed at 21 and 42 DOA. Serum samples were obtained by collecting a blood sample from the jugular vein in several 2 mL Eppendorf tubes for incubating at 37 °C for 2 h and then centrifuging at 2500g for 10 min. The resultant serum samples were stored in Eppendorf tubes at –70 °C until further analysis. In addition, 2 mL blood samples in EDTA-treated Vacutainer tubes were kept on ice and provided for immediate analysis of lymphocyte proliferation (only at 42 DOA). The broilers were then euthanized by cervical dislocation. The carcasses were subsequently opened, and the livers were removed aseptically. The liver tissues were collected into two 2 mL Eppendorf tubes wrapped with aluminum foil, were immediately frozen in liquid N2, and were then stored at –70 °C until further analysis. The thymus, spleen, and BF were collected. Adherent fat was removed, and the tissues were weighed. Immune organ index for thymus index (TI, thymus mass/body Published by NRC Research Press

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Table 1. Composition and nutrient levels of basal diet (dry matter basis).

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Item Raw material Corn Soybean (44.2% crude protein) Colza oil DL-Methionine Dicalcium phosphate Limestone Salt Vitamin Premix* Choline Mineral Premix† Total

51.64% 39.60% 4.30% 0.20% 1.85% 1.30% 0.40% 0.03% 0.18% 0.50% 100.00%

Nutrient level‡ CP DE Met Lys Thr Ca TP

23.93% 16.00 MJ/kg 0.52% 1.03% 0.77% 1.08% 0.77%

*Vitamin Premix provided the following per kilogram of basal diet: vitamin A, 50 000 IU; vitamin D-3, 10 000 IU; vitamin E, 25 IU; vitamin K, 35 mg; vitamin B-1, 2 mg; vitamin B-2, 16 mg; vitamin B-6, 6 mg; vitamin B-12, 0.03 mg; nicotinic, 25 mg; vitamin B-3, 25 mg; folic acid, 0.5 mg. †Mineral Premix provided the following per kilogram of basal diet: Fe (as ferrous sulfate), 80.00 mg; Cu (as copper sulfate), 8.00 mg; Mn (as manganese sulfate), 60.00 mg; Zn (as zinc sulfate), 40.00 mg; Se (as sodium selenite), 0.15 mg; I (as potassium iodate), 0.35 mg. ‡Nutrient levels were calculated by analysis. CP, crude protein; DE, digestible energy; Met, methionine; Lys, lysine; Thr, threonine; Ca, calcium; TP, total phosphorous.

mass ratio, g/kg), spleen index (spleen mass/body mass ratio, g/kg), and BF index (BF mass/body mass ratio, g/kg) were determined. Three broilers per treatment were selected from 10 broilers selected, and intestinal tracts were removed aseptically. The cecal contents were collected into two 2 mL Eppendorf tubes and were immediately stored at –70 °C until further analysis. All samples were analysed within a month of collection. Peripheral lymphocyte proliferation Peripheral lymphocyte proliferation in response to medium alone (control) or concanavalin A (20 ␮g/mL, Sigma Chemicals) was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL, Amersco) as described by Hung et al. (2010). Light absorbance at 570 nm was measured with an ELISA microplate reader (Thermo Electron Corporation, USA). The stimulation index was calculated as the ratio of the mean optical density value of mitogen-stimulated cells divided by the OD570 value of medium alone-stimulated (control) cells. Serum total IgG concentration Serum total IgG concentration, measured as a nonspecific response, was determined in appropriately diluted samples by a sandwich ELISA using microtiter plates and a chicken-specific IgG ELISA quantitation kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The ELISA procedure was carried out according to the protocol of the manufacturer, and the absorbance at 450 nm was determined using an ELISA microplate reader. The concentration of IgG was determined using standard curves constructed from respective Ig standards run on the assay microtiter plate and millilitres of serum. The linearity of the standard IgG was calculated using Excel, and found to be R2 = 0.98.

The levels of the cytokines interleukin-2 (IL-2) and IL-6 The concentrations of cytokines IL-2 and IL-6 in serum samples were determined with commercially available chicken cytokine RIA kits (North Institute of Biological Technology, Beijing, China), according to the manufacturer’s protocol. In each assay, a control recombinant chicken cytokine was diluted over the recommended detection range to generate a standard curve, and the linearity was calculated using Excel to be R2 = 0.99. Sample concentrations were interpolated from the standard curve. Antioxidant status determination Catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in serum and liver were determined using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute), following the manufacturer’s instructions. Specifically, CAT activity was measured via a colorimetric method and absorbance measurement at 405 nm; GSH-Px activity via a 5,5=-dithiobis-p-nitrobenzoic acid method and absorbance measurement at 412 nm; SOD activity via an auto-oxidation of hydroxylamine method and absorbance measurement at 450 nm; MDA content via a thiobarbituric acid method and absorbance measurement at 532 nm. Ten percent liver homogenates were obtained by homogenizing frozen liver tissue in ice-cold isotonic sterile physiological saline. The homogenates were then centrifuged (2500g or 3000g for 10 min) at 4 °C, and the protein concentration was determined using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute). Enzyme activity in liver was expressed as nanomoles per milligram of protein of the sample. The absorbance was determined using an ELISA microplate reader. Bacterial enumeration in cecal contents Deep frozen cecal contents were thawed for 10 min. Samples were immediately weighed and serially diluted up to 107 in sterile physiological saline. Dilutions were subsequently plated on duplicate selective agar media for enumeration of bacteria, including total aerobes, Escherichia coli, Enterococcus spp., total anaerobes, Lactobacillus spp., and Bifidobacterium spp. The appropriate selective agar media, incubation conditions and period, and colonies identification were in accordance with that described by Giannenas et al. (2012). Anaerobic incubation was achieved under anaerobic atmosphere (80% N2, 15% CO2, and 5% H2) without agitation. Results were expressed as base-10 logarithm colony-forming units per gram (wet mass) of cecal content (cfu/g). Polymerase chain reaction – denaturing gradient gel electrophoresis (PCR–DGGE) analysis Total bacteria DNA was isolated from all 200 mg of cecal contents using a previously described phenol extraction method (Tsai and Olsen 1992; Wilson and Blitchington 1996). The method of 16S rDNA amplification of the variable V3 region was combined with methods as described by Walter et al. (2001). The V3 region of the 16S rDNA gene (positions 339–539 in the E. coli gene) of bacteria was amplified by using primers HDA1-GC (5=-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAGT-3=) and HDA2 (5=-GTATTACCGCGGCTGCTGGCAC-3=). Each DNA sample was standardized to 20 ng/␮L and then amplified using primers specific for conserved sequences flanking the variable V3 region of 16S rDNA. The reaction mixture and amplification program were the same as described previously (Ni et al. 2009). After visual confirmation of the PCR products with 1.0% agarose gel electrophoresis, DGGE was performed using the Bio-Rad D-code system (Hercules, California). To separate PCR fragments, 35%⬃65% linear DNA-denaturing gradients (100% denaturant is equivalent to 7 mol/L urea and 40% deionized formamide) were formed in 8% polyacrylamide gels using a Bio-Rad Gradient Former. Bacterial V3 16S PCR products were loaded in each lane, and Published by NRC Research Press

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Table 2. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on daily body mass gain (BMG), daily feed intake (FI), and feed conversion ratio (FCR) of broilers. Treatment*

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A (LP–/BS–)

BS B (LP–/BS+)

1–21 DOA 22–42 DOA 1–42 DOA

Daily BMG (g) 33.33a 35.66b 79.30 86.40 56.87 61.65

1–21 DOA 22–42 DOA 1–42 DOA

Daily FI (g) 52.43 157.23 106.11

1–21 DOA 22–42 DOA 1–42 DOA

FCR (g/g) 1.57a 1.99 1.87

53.13 167.09 111.50 1.49b 1.94 1.81

C (LP+/BS–) 34.84b 82.10 59.04 50.42 153.51 103.22 1.45b 1.87 1.75

D (LP+/BS+) 35.20b 81.27 58.79 51.71 160.13 107.24 1.47b 1.97 1.82

SE

LP

BS–

BS+

Main effect (P value)

LP–

LP+

BS

LP

BS × LP

0.31 2.66 1.38

34.09 80.70 57.96

35.43 83.84 60.22

34.49 82.86 59.26

35.02 81.68 58.92

0.006 0.308 0.172

0.186 0.695 0.826

0.026 0.207 0.134

0.88 2.01 1.08

51.43 155.37 104.67

52.42 163.61 109.37

52.78 162.16 108.81

51.06 156.82 105.23

0.356 0.006 0.005

0.128 0.045 0.015

0.778 0.491 0.571

0.02 0.07 0.05

1.51 1.94 1.81

1.48 1.96 1.82

1.53 1.97 1.84

1.46 1.93 1.79

0.242 0.799 0.902

0.012 0.584 0.338

0.044 0.343 0.239

Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

Table 3. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on thymus index (TI), spleen index (BI), and bursa of Fabricius index (BFI) of broilers. Treatment*

BS

LP

Main effect (P value)

A (LP–/BS–)

B (LP–/BS+)

C (LP+/BS–)

D (LP+/BS+)

SE

BS–

BS+

LP–

LP+

BS

LP

BS × LP

21 DOA 42 DOA

TI (g/kg) 3.82 3.90a

4.56 4.69b

4.45 4.40ab

4.12 4.26ab

0.32 0.17

4.13 4.18

4.34 4.48

4.19 4.35

4.28 4.33

0.108 0.047

0.570 0.735

0.055 0.016

21 DOA 42 DOA

SI (g/kg) 0.76 1.01

0.78 1.01

0.80 1.22

0.84 1.03

0.09 0.12

0.78 1.13

0.81 1.02

0.77 1.01

0.82 1.13

0.905 0.435

0.806 0.346

0.650 0.420

21 DOA 42 DOA

BFI (g/kg) 1.97 1.70

1.84 1.71

1.98 1.54

2.23 1.58

0.16 0.10

1.97 1.63

2.03 1.64

1.91 1.71

2.10 1.56

0.288 0.982

0.090 0.274

0.952 0.630

Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

electrophoresis was performed at 60 °C at 100 V for 16–18 h. After electrophoresis, gels were silver-stained (Van Orsouw et al. 1997) and scanned using a Bio-Rad GS-800 Calibrated Imaging Densitometer. Each individual amplicon was then visualized as a distinct band representing at least one bacterial species on the gel. The fingerprints of DGGE profiles and the construction of dendrograms were carried out with Quantity One analysis software version 4.6.2 (Bio-Rad) and UPGMA (unweighted pair group mean average) algorithm. Statistical analyses Statistical analysis of all alphanumeric data was completed with statistical package of SPSS version 19 for Windows (SPSS Inc., Chicago, Illinois, USA). Any significant effects and interactions of BS and LP were analyzed as a 2 × 2 factorial arrangement by 2-way ANOVA. Duncan’s multiple-range test was used for multiple comparisons when the interaction between BS and LP was significant. All statements of significance were based on P ≤ 0.05. All statistical calculations were performed with Excel 2013 (Microsoft, Redmond, Washington, USA).

Results Production performance The interaction between LP and BS significantly affected daily BMG (P = 0.026) and FCR (P = 0.044) in the starter period (1–21 DOA).

In the starter period (1–21 DOA), broilers administered LP and (or) BS (LP–/BS+, LP+/BS–, LP+/BS+) had significantly higher daily BMG and lower FCR than the control (LP–/BS–) but did not differ from one another (Table 2). The interaction between BS and LP had no significant effect on daily FI in either phase or the overall period of the experiment. However, in the grower period (22–42 DOA) and overall period (1–42 DOA), BS immunization increased daily FI (P = 0.006 and 0.005, respectively), and LP supplementation decreased daily FI (P = 0.045 and 0.015, respectively). There were no significant effects of BS and LP on daily FI in the starter period (1–21 DOA), daily BMG and FCR in the grower period (22–42 DOA), or overall period (1–42 DOA). Immune organ index The interaction between BS and LP significantly affected TI at 42 DOA (P = 0.016). The TI of broilers immunized with BS alone (LP–/BS+) was significantly higher than that of broilers in the control (LP–/BS–) at 42 DOA (Table 3). However, there were no significant effects of BS and LP on TI at 21 DOA and on stimulation index and BF index at both 21 and 42 DOA. Peripheral lymphocyte proliferation The interaction between BS and LP significantly affected peripheral lymphocyte proliferation at 42 DOA (P = 0.000) (Table 4). Peripheral lymphocyte proliferation induced by the T-cell mitogen Published by NRC Research Press

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Table 4. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on stimulation index (SI) of peripheral lymphocyte proliferation of broilers. Treatment*

42 DOA

BS

LP

Main effect (P value)

A (LP–/BS–)

B (LP–/BS+)

C (LP+/BS–)

D (LP+/BS+)

SE

BS–

BS+

LP–

LP+

BS

LP

BS × LP

SI (%) 103.38a

117.92b

147.01c

118.70b

3.92

125.20

118.31

110.65

132.86

0.148

0.000

0.000

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Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

Table 5. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on total immunoglobulin G (IgG) concentration in serum of broilers. Treatment* A (LP–/BS–) 21 DOA 42 DOA

BS B (LP–/BS+)

Total IgG concn. (␮g/mL) 1.319 1.508 2.189 2.478

LP

Main effect (P value)

C (LP+/BS–)

D (LP+/BS+)

SE

BS–

BS+

LP–

LP+

BS

LP

BS × LP

1.267 1.690

1.863 2.895

0.141 0.184

1.29 1.94

1.67 2.71

1.41 2.33

1.53 2.36

0.045 0.005

0.602 0.946

0.075 0.056

Note: DOA, day of age. *Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

Table 6. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on concentrations of interleukin 2 (IL-2) and IL-6 in serum of broilers. Treatment* A (LP–/BS–)

BS B (LP–/BS+)

21 DOA 42 DOA

IL-2 concn. (ng/mL) 14.13a 16.40b 13.86 14.03

21 DOA 42 DOA

IL-6 concn. (pg/mL) 156.58 194.73 218.43 258.58

C (LP+/BS–) 17.36b 15.89 164.97 250.93

D (LP+/BS+) 15.53ab 16.18 210.08 281.81

SE

BS–

LP BS+

LP–

Main effect (P value) LP+

BS

LP

BS × LP

0.67 0.87

15.74 14.88

15.97 15.10

15.26 13.95

16.45 16.04

0.749 0.804

0.106 0.030

0.009 0.946

15.31 13.09

160.78 234.68

202.40 270.20

175.65 238.51

187.53 266.37

0.019 0.024

0.469 0.068

0.831 0.749

Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

concanavalin A was greater in broilers supplemented with LP and (or) BS immunized (LP–/BS+, LP+/BS–, LP+/BS+) compared with the control (LP–/BS–). The highest level of peripheral lymphocyte proliferation occurred in the treatment LP administered alone (LP+/BS–) (P ≤ 0.05). Serum total IgG concentration The interaction between BS and LP had no significant effect on total IgG concentrations in the serum at both 21 and 42 DOA. However, BS immunization increased total IgG concentrations in the serum at both 21 and 42 DOA (P = 0.045 and 0.005, respectively) (Table 5). The concentrations of the cytokines IL-2 and IL-6 The interaction between BS and LP significantly affected IL-2 concentration at 21 DOA (P = 0.009). The IL-2 concentrations of broilers administered BS alone or LP alone (LP–/BS+, LP+/BS–) were significantly higher than that of broilers in the control (LP–/BS–) at 21 DOA but did not differ significantly from each other (Table 6). The interaction between BS and LP had no significant effect on IL-2 concentration at 42 DOA nor on IL-6 concentration at both 21 and 42 DOA. However, LP supplementation increased IL-2 concentration (P = 0.030) of broilers at 42 DOA, and BS immunization increased IL-6 concentration of broilers at both 21 and 42 DOA (P = 0.019 and P = 0.024, respectively).

Antioxidant status in serum and liver Both BS and LP alone and in combination had no significant effect on all antioxidant parameters in the serum and liver at both 21 and 42 DOA, except for MDA content in the liver at 21 DOA (P = 0.034) (Table 7). Broilers administered LP and (or) BS (LP–/BS+, LP+/BS–, LP+/BS+) had a significantly lower liver MDA content than the control (LP–/BS–) but did not differ significantly among each other. However, BS immunization increased CAT activities in the serum (P = 0.017 and 0.004, respectively) and liver (P = 0.013 and 0.042, respectively) at both 21 and 42 DOA and decreased serum MDA content (P = 0.038) at 42 DOA. LP supplementation increased GSH-Px activities in the serum (P = 0.005) and liver (P = 0.007) at 21 DOA and liver CAT activities (P = 0.003 and 0.017, respectively) at both 21 and 42 DOA and decreased serum MDA content (P = 0.000) at 42 DOA. There were no significant effects of BS and LP on the serum MDA content at 21 DOA, on the liver MDA content and the GSH-Px activities in serum and liver at 42 DOA, and on the activities of SOD in the serum and liver at both 21 and 42 DOA. Intestinal microflora composition Both BS and LP alone and in combination had no significant effect on all bacterial counts at both 21 and 42 DOA, except for E. coli count at 42 DOA (P = 0.019). Broilers administered LP and (or) BS (LP–/BS+, LP+/BS–, LP+/BS+) had a significantly lower E. coli count than the control (LP–/BS–) but did not significantly differ among each other (Table 8). LP supplementation decreased total Published by NRC Research Press

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Table 7. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in serum and liver of broilers. Treatment* A (LP–/BS–)

BS B (LP–/BS+)

C (LP+/BS–)

D (LP+/BS+)

SE

LP

BS–

BS+

Main effect (P value)

LP–

LP+

BS

LP

BS × LP

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Serum 21 DOA 42 DOA

Antioxidant status CAT activity (U/mL) 1.45 2.12 1.97 1.34 1.90 1.66

21 DOA 42 DOA

SOD activity (U/mL) 87.45 86.63 151.30 164.67

21 DOA 42 DOA

GSH-Px activity (U/mL) 500.09 459.52 684.29 700.03

21 DOA 42 DOA

MDA content (nmol/mL) 6.53 6.76 7.78 5.42

2.81 2.22

0.28 0.16

1.74 1.50

2.43 2.06

1.82 1.62

2.34 1.94

0.017 0.004

0.056 0.070

0.234 0.995

88.65 155.13

86.63 147.41

3.13 10.77

88.05 153.21

86.63 156.04

87.04 157.99

87.64 151.27

0.175 0.551

0.904 0.568

0.608 0.088

552.59 707.47

622.37 716.35

36.08 49.24

527.09 695.88

540.95 708.19

479.81 693.03

588.23 712.40

0.156 0.678

0.005 0.215

0.250 0.317

5.53 2.40

3.21 2.09

0.40 0.37

6.16 5.09

5.43 4.17

6.65 6.46

4.37 2.27

0.256 0.038

0.061 0.000

0.085 0.120

21 DOA 42 DOA

Antioxidant status CAT activity (U/mg protein) 2.88 5.15 6.15 7.67 6.34 6.93 6.86 11.73

0.54 0.76

4.74 6.60

6.23 8.99

4.18 6.68

6.80 9.30

0.013 0.042

0.003 0.017

0.307 0.176

21 DOA 42 DOA

SOD activity (U/mg protein) 103.48 105.01 117.59 92.25 95.48 96.51

105.23 100.75

4.79 4.13

110.54 94.38

105.12 98.11

104.25 93.86

111.41 98.63

0.309 0.439

0.186 0.327

0.198 0.915

21 DOA 42 DOA

GSH-Px activity (U/mg protein) 23.39 20.88 28.57 24.38 27.52 26.83

29.97 32.10

1.23 1.94

25.98 25.61

25.43 29.81

22.14 25.95

29.27 29.47

0.940 0.140

0.007 0.200

0.991 0.980

21 DOA 42 DOA

MDA content (nmol/mg protein) 0.91a 0.54b 0.60b 1.09 1.00 0.92

0.05 0.12

0.76 1.01

0.62 1.05

0.73 1.05

0.65 1.01

0.054 0.095

0.710 0.587

0.034 0.644

Liver

0.69b 1.09

Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

Table 8. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on intestinal microflora composition in cecal contents of broilers. Treatment* A (LP–/BS–)

BS B (LP–/BS+)

C (LP+/BS–)

D (LP+/BS+)

SE

BS–

LP BS+

LP–

Main effect (P value) LP+

BS

LP

BS × LP

21 DOA 42 DOA

Total aerobes (log10 cfu/g) 9.38 9.00 8.75 8.69

8.45 8.15

8.60 8.51

0.21 0.26

8.92 8.45

8.80 8.60

9.19 8.72

8.53 8.33

0.628 0.660

0.024 0.259

0.301 0.532

21 DOA 42 DOA

Escherichia coli (log10 cfu/g) 7.73 7.40 7.36a 6.86ab

6.86 6.46b

7.22 6.86ab

0.18 0.15

7.30 6.91

7.31 6.86

7.57 7.11

7.04 6.66

0.951 0.761

0.036 0.019

0.139 0.019

21 DOA 42 DOA

Enterococcus spp. (log10 cfu/g) 7.54 7.35 7.27 7.43 7.44 7.05

7.33 7.39

0.33 0.15

7.40 7.24

7.34 7.42

7.45 7.44

7.30 7.22

0.871 0.317

0.692 0.233

0.739 0.362

21 DOA 42 DOA

Total anaerobes (log10 cfu/g) 9.66 10.05 10.38 10.09 10.48 10.42

10.29 10.55

0.14 0.22

10.02 10.26

10.17 10.52

9.86 10.29

10.33 10.49

0.357 0.273

0.017 0.398

0.166 0.570

21 DOA 42 DOA

Lactobacillus spp. (log10 cfu/g) 8.62 8.99 9.53 8.93 9.00 9.72

9.53 9.68

0.18 0.20

9.07 9.32

9.26 9.34

8.80 8.97

9.53 9.70

0.334 0.930

0.004 0.007

0.342 0.794

21 DOA 42 DOA

Bifidobacterium spp. (log10 cfu/g) 8.51 9.13 9.42 8.79 9.07 9.87

9.55 9.72

0.19 0.16

8.96 9.33

9.34 9.40

8.82 8.93

9.49 9.80

0.095 0.711

0.010 0.001

0.251 0.254

Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized. Published by NRC Research Press

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Fig. 1. PCR–DGGE profiles combined with dendrogram and the percentage coefficient (bar) of the V3 region gene of 16S rDNA of microflora in cecal contents of broilers at 21 days of age (A) and 42 days of age (B). Three representative samples from each treatment are shown. Treatments: A (LP–/BS–), both Lactobacillus plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

aerobes (P = 0.024) and E. coli (P = 0.036) counts at 21 DOA, increased total anaerobes (P = 0.017) count at 21 DOA, increased Lactobacillus spp. (P = 0.004 and 0.007, respectively) and Bifidobacterium spp. (P = 0.010 and 0.001, respectively) counts at both 21 and 42 DOA. There were no significant effects of BS and LP on the total aerobes and total anaerobes counts at 42 DOA and on the Enterococcus spp. counts at both 21 and 42 DOA. Intestinal microflora diversity Cluster analysis of universal bacterial DGGE profiles from cecal contents of broilers at 21 and 42 DOA is presented in Figs. 1A and 1B, respectively. The dendrograms of intestinal diversity are divided into 2 main sections: section 1 corresponds to the treatments supplemented with LP in the diet (samples C1–D3), section 2 corresponds to treatments not supplemented with LP (samples A1–B3) (0.58% similarity in Fig. 1A and 0.59% similarity in Fig. 1B). In Fig. 1A section 1, the structural similarity of C2 and C3, D1 and D3 was higher (0.78 and 0.77 similarity, respectively), whereas the

similarity between these 2 groups was 0.68%. In section 2, there was no difference in similarity among samples A1–B3. In Fig. 1B section 1, the structural similarity of D1 and D2 was highest (0.91% similarity) and had the lowest similarity with C1 (0.66% similarity). In section 2, the structural similarity of B1 and B2 was highest (0.92% similarity) and had the lowest similarity with group A1 and A3 (0.78% similarity). Table 9 presents the mean numbers of 16S rDNA PCR–DGGE bands (amplicons) in each treatment. The interaction between BS and LP significantly affected the number of bands at 21 DOA (P = 0.001). Broilers immunized with BS alone (LP–/BS+) had a significantly greater number of bands than broilers with other treatments; the other treatments (LP–/BS–, LP+/BS–, LP+/BS+) did not differ significantly from each other. The interaction between BS and LP had no significant effect on the number of bands at 42 DOA. However, BS immunization increased the number of bands at 42 DOA (P = 0.037). Published by NRC Research Press

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Table 9. Effects of Lactobacillus plantarum (LP) dietary supplementation and bursin (BS) immunization on band numbers in PCR–DGGE profiles of the V3 region gene of 16S rDNA of microflora in cecal contents of broilers. Treatment* A (LP–/BS–) 21 DOA 42 DOA

BS B (LP–/BS+)

No. of bands 26.67a 35.33b 27.00 33.33

LP

Main effect (P value)

C (LP+/BS–)

D (LP+/BS+)

SE

BS–

BS+

LP–

LP+

BS

LP

BS × LP

26.67a 27.00

25.33a 29.00

0.85 1.65

26.67 27.00

30.33 31.17

31.00 30.17

26.00 28.00

0.008 0.037

0.001 0.230

0.001 0.230

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Note: DOA, day of age. *Means within rows followed by different letters are significantly different (P ≤ 0.05). Treatments: A (LP–/BS–), both L. plantarum nonsupplemented and bursin nonimmunized; B (LP–/BS+), L. plantarum nonsupplemented and bursin immunized; C (LP+/BS–), L. plantarum supplemented and bursin nonimmunized; D (LP+/BS+), L. plantarum supplemented and bursin immunized.

Discussion Lactobacillus enhance production performance and immune responses of broilers by modulating intestinal microflora and by developing immunity of the intestinal tract. BS isolated from BF has demonstrated important immune function. The synergy of LP dietary supplementation and BS immunization in broilers were evaluated in this study. Our results indicated that broilers administered BS and (or) LP had improved production performance with higher daily BMG and lower FCR in the starter period (1–21 DOA). Although there was no previous research about the effect of BS immunization on production performance in broilers, in the present study, the effect of LP supplementation on the production performance of broilers was consistent with the results of a series of earlier studies (Yeo and Kim 1997; Zulkifli et al. 2000; Kalavathy et al. 2008). The nonstatistical differences of FCR values during the grower period could be due to the increased daily FI, which was higher in broilers immunized with BS in the grower period (22– 42 DOA) and overall period (1–42 DOA), meanwhile LP supplementation decreased daily FI of broilers. Zulkifli et al. (2000) and Kalavathy et al. (2008) observed that better feed efficiency was achieved in broilers fed an LP diet than a control diet from 1–21 DOA, whereas more feed was consumed by the broilers because the broilers were subjected to cyclic heat stress from 22– 42 DOA. However, in Huang’s research, he attributed significantly higher BMG and lower FCR of broilers in the second week to the starter diet feed (Huang et al. 2004). For this reason, our broilers were fed a single basal diet over the entire trial, and similar results were observed. This work investigated the effects of LP and BS on cellular immunity and humoral immunity, respectively. IL-6, named B-cell stimulating cytokine, can promote B lymphocyte proliferation, differentiation, and induction of Ig production. The level of IL-6 can reflect the body’s humoral immunity. BS is generally known as the hormone that selectively influences the differentiation of B cells but not T cells in poultry (Audhya et al. 1986; Yuko et al. 2001). The effect of BS immunization on the humoral immunity of broilers was reflected in our data, with a significantly higher concentration of serum total IgG and IL-6 concentration. Meanwhile, broilers administered LP and (or) BS showed higher levels of lymphocyte proliferation and IL-2 concentration. The level of lymphocyte proliferation in response to concanavalin A is a significant method for measuring cellular immunity (Lafuente et al. 2003). IL-2 is produced by lectin- or antigen-activated T cells (Li et al. 2004). It is a crucial cytokine for survival of T lymphocytes and proliferation of activated T lymphocyte. The level of IL-2 can reflect a body’s cellular immunity. Our results indicated that LP and (or) BS significantly enhanced cellular immunity of broilers. The results of broilers administered LP agreed with those of Won et al. (2011). Additionally, some studies reported that a high dose of chicken BF extract or BS could influence the differentiation of T lymphocytes (Brand et al. 1976; Guo et al. 2006). It might be that BS like BP5, a peptide isolated from chicken BF, promotes lymphocyte proliferation by activating B cells directly and T cells indirectly (Hauge et al. 2007; Li et al. 2011).

To demonstrate the potential of probiotics to enhance the capacity of the humoral immune system of broilers, many studies measure the humoral immune responses against specific model antigens. Even so, there have been some studies with inconsistent results (Huang et al. 2004; Haghighi et al. 2005). In this work, we aimed to get the humoral immune status at a systemic level but not the immune response capacity against a specific antigen. However, our results showed that LP supplementation had no effect on serum IgG concentration, which is in agreement with Mountzouris et al. (2010). The reason might be that selected immunologic lactic acid bacteria stimulated the T-cell immune system via a toll-like receptor in the gut, resulting in an enhanced intestinal cell-mediated mucosal immunity, but did not affect the B-cell-related immune system (Dalloul et al. 2003, 2005; Sato et al. 2009). The immune organ index reflects the ability to provide lymphoid cells during immune response (Heckert et al. 2002). The roles of BS and LP on immune organ index revealed that BS immunization resulted in markedly increased TI at 42 DOA in this study. BS, isolated from BF, has been shown to promote the development of the BF and BF follicles (Audhya et al. 1986; Yuko et al. 2001). However, no difference was found in BF index of broilers immunized with BS in our study. There also were different effects on immune organ mass of broilers given the Lactobacillus diet in some previous studies (Jin et al. 1998a; Li et al. 2009). The discrepancy among these studies may be attributed to several factors, such as broiler breeds and physiological stages, diet compositions, and LP and BS administration levels. Overall, the results showed that LP dietary supplementation combined with BS immunization displayed synergistic modulation effects on cellular immunity, and BS immunization enhanced humoral immunity of broilers fed the LP diet. Antioxidant enzymes are able to eliminate reactive oxygen species and lipid peroxidation products, as a result of protecting cells and tissues from oxidative damage. Antioxidant enzymes, including CAT, SOD, and GSH-Px, are important buffers in the interception and degradation of superoxide anion and hydrogen peroxide and protect against oxidative stress (Bhatia et al. 2003; Wang et al. 2008b). As an end product of lipid peroxidation, MDA can react with biomolecules and exert cytotoxic and genotoxic effects. It also causes mutagenic lesions that may be involved in the pathology of various diseases. MDA content is often used as an indicator of oxidative damage and ageing in an organism (Spiteller 2001). Some Lactobacillus strains had been found with antioxidant activities and biological functions in different animals and in humans (Kullisaar et al. 2002; Wang et al. 2009; Zhang et al. 2010; Kang et al. 2012; Li et al. 2012). Our data indicated that LP enhanced antioxidant capacities in serum and liver of broilers by increasing GSH-Px and CAT activities and by decreasing MDA contents. Our study is the first to show the antioxidant function of BS immunization in broilers by increasing CAT activity and decreasing MDA contents in serum and liver. Although there was only a synergistic effect of LP and BS on MDA content at 21 DOA, LP dietary supplePublished by NRC Research Press

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Shen et al.

mentation and BS immunization could contribute to a host’s health through their antioxidative effects. It is generally accepted that the intestinal microflora contributes to intestinal function through competitive exclusion and colonization resistance or bacterial interference (Nurmi and Rantala 1973). Traditionally, the cecal microflora has been analyzed using culture-dependent methods. That Lactobacillus fortify the intestinal microflora composition was evidenced by the significant increases of beneficial bacteria counts (Lactobacillus spp. and Bifidobacterium spp.) and decreases of harmful bacteria counts (E. coli) in the intestinal contents of broilers (Yu et al. 2008; Xie et al. 2011). The similar results at 21 and 42 DOA were observed in this study. However, the similar bacterial counts of broilers with or without BS immunization in this study indicated that BS immunization had no effect on intestinal microflora composition. Meanwhile, the higher resolution molecular technique PCR– DGGE of the V3 region of 16S rDNA had compared microbial populations and their diversity. PCR–DGGE profiles combined with dendrogram showed clustering according to supplementation of LP. It indicated the addition of LP to the diet changed intestinal microflora composition significantly over the period of the study. Netherwood et al. (1999) showed that digestive bacterial populations could be influenced by diet. Fuentes et al. (2008) also demonstrated that group-specific DGGE profiles revealed clear separation of animals, depending on feeding of lactobacilli strains. However, there was no grouping by treatments immunized with BS. It indicated that BS immunization maintained stable influence on the structure of intestinal microflora of broilers, which is consistent with the results of BS immunization on intestinal bacterial counts. The main reason for higher similarity in the BS immunization treatments (0.91% similarity between D1 and D2, 0.92% similarity between B1 and B2) at 42 DOA compared with the nonimmunization treatments might be that BS immunization enriched intestinal microflora diversity. This supposal was supported by the mean numbers of 16S rDNA PCR–DGGE bands (amplicons) in each treatment. The data had shown BS immunization increased the diversity of intestinal microflora of broilers at 21 and 42 DOA. Since the addition of LP lowered pH in the tract and thus might have selected for the most aciduric members, which release bacteriocin or bacteriocin-like substances that negatively affect other, potentially pathogenic microbial species (Klaehammer 1993; Corr et al. 2007), LP aimed at the improvement of gastrointestinal microbial balance, increased the diversity of gut Lactobacillus genus, while overall bacterial diversity remained largely unaffected (Lan et al. 2004; Saxelin et al. 2005; Fuentes et al. 2008). Overall, the results in this work showed that LP dietary supplementation combined with BS immunization displayed synergistic modulation effects on the production performance in the starter period; IL-2 concentration and liver MDA content at 21 DOA; and TI, peripheral lymphocyte proliferation, and E. coli counts at 42 DOA. However, the addition of LP in the diet of BS-immunized broilers promoted production performance, enhanced immune responses from cellular immunity aspect and humoral immunity aspect, improved antioxidant capacities including CAT activity, GSH-Px activity and MDA content, and fortified intestinal microflora by balancing intestinal microflora composition and enriching intestinal microflora diversity.

Acknowledgement The present study was supported by the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry, Program for Changjiang Scholars and Innovative Research Team of the University of China (ITT0848) and by the International Cooperative Project of Science and Technology Bureau of Sichuan Province (2013HH0055).

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